CN111925925A - Integrated diagnostic kit and application thereof - Google Patents
Integrated diagnostic kit and application thereof Download PDFInfo
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- CN111925925A CN111925925A CN202010826506.3A CN202010826506A CN111925925A CN 111925925 A CN111925925 A CN 111925925A CN 202010826506 A CN202010826506 A CN 202010826506A CN 111925925 A CN111925925 A CN 111925925A
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- 238000009007 Diagnostic Kit Methods 0.000 title claims abstract description 18
- 238000003860 storage Methods 0.000 claims abstract description 122
- 238000004140 cleaning Methods 0.000 claims abstract description 119
- 239000007788 liquid Substances 0.000 claims abstract description 73
- 230000003321 amplification Effects 0.000 claims abstract description 56
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 56
- 239000003480 eluent Substances 0.000 claims abstract description 40
- 230000009089 cytolysis Effects 0.000 claims abstract description 39
- 238000005336 cracking Methods 0.000 claims abstract description 36
- 239000011324 bead Substances 0.000 claims abstract description 30
- 239000002699 waste material Substances 0.000 claims abstract description 30
- 238000010828 elution Methods 0.000 claims abstract description 29
- 238000001514 detection method Methods 0.000 claims abstract description 13
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
- 230000009471 action Effects 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
- 238000000197 pyrolysis Methods 0.000 claims description 8
- 238000012408 PCR amplification Methods 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 6
- 239000006166 lysate Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000012197 amplification kit Methods 0.000 claims 4
- 241000700605 Viruses Species 0.000 abstract description 3
- 244000052616 bacterial pathogen Species 0.000 abstract description 3
- 208000024891 symptom Diseases 0.000 abstract description 3
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 10
- 230000008569 process Effects 0.000 description 8
- 238000003752 polymerase chain reaction Methods 0.000 description 7
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000007403 mPCR Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000012502 diagnostic product Substances 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012123 point-of-care testing Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses an integrated diagnostic kit and application thereof, and relates to the technical field of biomedical detection and diagnosis. The body of the kit is in a disc shape and comprises a sample adding groove, a cleaning solution storage groove, a lysis solution storage groove, an eluent storage groove, an oil storage groove, a lysis cleaning groove, an elution groove, an amplification groove and a waste liquid storage groove; the liquid storage area comprises a cracking liquid tank, a cleaning liquid tank, an elution liquid tank and an oil tank; the sample loading slot is positioned in the middle of the disc, the liquid storage area is concentrically distributed around the sample loading slot, and the amplification slot is positioned at the edge of the kit. According to the invention, by designing a disc structure, a matching motor drives a kit to rotate to generate centrifugal force, and under the action of magnetic force and PCR magnetic beads, the lysis cleaning, elution and amplification of sample genes are completed, and finally, the result is obtained by detection. The invention adopts the disc structure to shorten the detection time to 20 to 30 minutes, and can simultaneously detect a plurality of germs and viruses causing symptoms.
Description
Technical Field
The invention relates to the technical field of biomedical detection and diagnosis, in particular to an integrated diagnostic kit and application thereof.
Background
In the field of infectious diseases, in order to control the spread of infection, it is necessary to rapidly derive the cause of the disease or whether a person is infected, and a very rapid and accurate molecular diagnostic product is required.
The FilmArray product from BioFire is a classic work that has been successfully commercialized at present, and employs nested multiplex PCR analysis techniques. The entire FilmArray reagent may be simply divided into two parts, an upper reservoir group and a lower reaction layer. The basic structure of such a liquid storage tube set and the principle and process of sucking liquid by vacuum are disclosed in patent US8409508, and a single liquid storage tube is attached to a flexible bag by means of hot plastic sealing, wherein the bottom of the liquid storage tube is communicated with a reaction tank in the flexible bag. The liquid storage tube wall has two openings, the opening on the upper side is the exhaust port, and the inside air is extracted to form the vacuum cavity when being used for liquid storage tube encapsulation, and the opening on the lower side is the reagent entry, generally is sealed state, and the reagent can be inhaled by the vacuum after puncturing.
Like other POCT products, the FilmArray also adopts a test instrument and a disposable test chip mode for detection, and compared with other disease detection chips on the market, the product has very obvious competitive advantages, can simultaneously detect various germs and viruses causing symptoms, finally locates the causes causing diseases, and mainly benefits from a multiple PCR technology.
However, the manufacturing process of the kit is very complex, which leads to high price of the FilmArray product, so that an integrated diagnostic kit needs to be designed in a targeted manner, and on the premise of meeting various detection requirements and not changing the product performance and convenience, the problem of process complexity is solved, and the product cost is reduced.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an integrated diagnostic kit, which solves the problem of process complexity and reduces the product cost on the premise of meeting various detection requirements and not changing the product performance and convenience.
In order to achieve the purpose, the invention provides an integrated diagnostic kit, which comprises a disc-shaped kit body, and a sample adding groove, a cleaning solution storage groove, a lysis solution storage groove, an eluent storage groove, an oil storage groove, a lysis cleaning groove, an elution groove, an amplification groove and a waste liquid storage groove which are distributed on the body;
wherein:
the sample loading slot is positioned in the middle of the kit, and the cleaning liquid storage tank, the lysis liquid storage tank, the eluent storage tank and the oil storage tank are concentrically arranged with the sample loading slot; the position of the amplification tank is close to the edge of the kit, and the cracking cleaning tank and the elution tank are close to the liquid storage area;
the sample loading groove is communicated with the cracking cleaning groove through a loading flow channel; the cracking cleaning tank is communicated with the waste liquid storage tank through a cleaning waste liquid flow channel; the cracking cleaning tank is communicated with the elution tank through a magnetic bead moving channel; the elution groove is communicated with the amplification groove through a capillary flow passage;
at least one of the cleaning solution storage tank, the cracking solution storage tank, the eluent storage tank and the oil storage tank is arranged;
the cleaning solution storage tank is provided with a cleaning solution overflow port, the cleaning solution overflow port is communicated with a cleaning solution flow channel, and the cleaning solution flow channel is communicated with the cracking cleaning tank;
the cracking liquid storage tank is provided with a cracking liquid overflow port which is communicated with the cracking cleaning tank;
the eluent storage tank is provided with an eluent overflow port which is communicated with the elution tank;
the oil storage tank is provided with an amplification flow channel which is convenient for oil to flow out, and the amplification flow channel is communicated with the amplification tank.
Further, the sample loading slot is round,
further, the sample loading slot is positioned in the center of the kit body;
furthermore, the cleaning solution storage tank, the lysis solution storage tank, the eluent storage tank and the oil storage tank are arranged around the sample loading slot and are fan-shaped;
furthermore, more than two cleaning solution storage tanks are provided, each cleaning solution storage tank is provided with an overflow port, the cleaning solution storage tanks are respectively communicated with a cleaning solution flow channel, and the cleaning solution flow channels are annular and are positioned on the outer walls of the cleaning solution storage tanks;
furthermore, the amplification grooves comprise more than two and are communicated in sequence; the amplification groove and the sample loading groove are concentrically arranged;
further, the waste liquid storage groove and the sample loading groove are arranged concentrically and are close to the edge of the kit;
further, the cleaning waste liquid flow channel is U-shaped or S-shaped;
further, the capillary flow channel is U-shaped or S-shaped.
It is still another object of the present invention to provide a method for detecting a target gene using an integrated diagnostic kit, comprising the steps of:
1. placing the kit on a matched workbench with a motor, adding a PCR sample into a sample adding groove, connecting the kit with the matched motor, adding a lysis solution into a lysis solution storage groove, starting the motor, synchronously rotating the kit, allowing the sample and the lysis solution to enter a lysis cleaning groove under the action of centrifugal force, adding PCR magnetic beads, and performing lysis reaction on the sample; after the reaction is finished, magnetic force is applied, and the centrifugal force of a motor centrifugally flows the reacted lysate into a waste liquid storage tank;
2. removing the magnetic force, adding cleaning solution into the cleaning solution storage tank, starting a motor, stopping the motor after the cleaning solution flows into the cracking cleaning tank, and cleaning under vibration and stirring; after the washing is finished, applying magnetic force, and enabling the aggregated magnetic beads to enter an elution tank along a magnetic bead channel; starting a motor, and centrifugally flowing the cleaning waste liquid into a waste liquid storage tank;
3. adding eluent into an eluent storage tank, rotating with a motor to enter an elution tank, removing magnetic force, stopping the motor, performing vibration stirring elution operation, adding magnetic force to remove magnetic beads after the operation is finished, starting the motor, and enabling the eluent to enter an amplification tank through a capillary flow channel;
4. and adding oil into the oil storage tank, entering the amplification tank along with the rotation of the motor, controlling the temperature-variable circulation of the cavity to carry out amplification reaction, and detecting a fluorescence signal of the amplification tank after the reaction is finished to obtain a detection result.
Preferably, the step 2 can be repeated twice or more as required;
further, in the step 4, a freeze-drying reagent required for PCR amplification is pre-stored in the amplification tank, and the freeze-drying reagent comprises primers required for an amplification reaction and other components required for PCR amplification;
further, the step 4 may be performed as many times as necessary for amplification.
The working principle of the integrated diagnostic kit provided by the invention is as follows: under the action of the rotating centrifugal force of the matching motor, corresponding reagents in the kit flow into a communicated area along with the overflow port and enter a cracking cleaning tank, an elution tank and an amplification tank respectively, PCR magnetic beads are adsorbed or released by virtue of magnetic force to complete cracking cleaning and elution, and finally amplification and reaction are carried out, and an amplified fluorescent signal is detected to obtain a detection result.
Compared with the prior art, the invention has the following beneficial effects:
1. the scheme of the invention adopts a disc structure, can solve the problem of process complexity, greatly reduces the cost of the product on the premise of not changing the performance and convenience of the product, and shortens the detection time to 20 to 30 minutes;
2. the kit is used for carrying out multiplex PCR amplification, and can simultaneously detect a plurality of pathogenic bacteria and viruses causing symptoms.
Drawings
FIG. 1 is a schematic view of an embodiment of the integrated diagnostic kit of the present invention.
FIG. 2 is a partially enlarged schematic view of one embodiment of the integrated diagnostic kit of the present invention.
In the figure: 1 sample adding groove, 2 cleaning solution storing groove, 3 cracking solution storing groove, 4 eluent storing groove, 5 oil storing groove, 6 cracking cleaning groove, 7 eluent groove, 8 amplification groove, 9 waste solution storing groove, 10 cleaning waste solution flow channel, 11 capillary flow channel, 12 cracking solution overflow port, 13 sample adding flow channel, 14 cleaning solution overflow port, 15 cleaning solution flow channel, 16 magnetic bead moving channel, 18 amplification flow channel, 19 reagent box body.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Examples
Referring to fig. 1 and 2 (fig. 2 is an enlarged view of a portion a in fig. 1), the present embodiment provides an integrated diagnostic kit, wherein a kit body (19) is disc-shaped and is distributed on the body: the sample adding device comprises a sample adding groove (1), a liquid storage area, a cracking cleaning groove (6), an elution groove (7), an amplification groove (8) and a waste liquid storage groove (9), wherein the liquid storage area is composed of a cleaning liquid storage groove (2), a cracking liquid storage groove (3), an eluent storage groove (4) and an oil storage groove (5);
the sample adding slot (1) is round and is positioned in the middle of the kit, and the central position is optimized; the liquid storage area is concentrically arranged around the sample loading slot (1); the amplification tank (8) and the waste liquid storage tank (9) are positioned close to the edge of the kit; at least one amplification groove (8) (5 in the embodiment and the attached drawings, sequentially communicated, concentric with the sample loading groove (1) and arranged in a fan shape, and the number of the amplification grooves can be actually adjusted according to needs); the waste liquid storage tank (9) and the sample loading tank (1) are concentrically arranged; the cracking cleaning tank (6) and the elution tank (7) are close to the liquid storage area;
a sample adding runner (13) is arranged between the sample adding slot (1) and the cracking cleaning tank (6), and when a sample is added into the sample adding slot (1), the sample can enter the cracking cleaning tank (6) through the sample adding runner (13); a cleaning waste liquid flow passage (10) is arranged between the cracking cleaning tank (6) and the waste liquid storage tank (9); a magnetic bead moving channel (16) is arranged between the cracking cleaning tank (6) and the elution tank (7); a capillary flow channel (11) is arranged between the elution tank (7) and the amplification tank (8); the cleaning waste liquid flow channel (10) and the capillary flow channel (11) are both U-shaped (the structures of the embodiment and the attached drawings are both U-shaped; one of the two can be designed into S-shaped or S-shaped according to actual needs);
the liquid storage area comprises at least one cleaning liquid storage tank (2), at least one lysis solution storage tank (3), at least one eluent storage tank (4) and at least one oil storage tank (5) (in the embodiment and the attached drawings, the cleaning liquid storage tanks are 4, the lysis solution storage tanks are 1, the eluent storage tanks are 1, and the oil storage tanks are 1, the number of the cleaning liquid storage tanks, the lysis solution storage tanks and the eluent storage tanks can be adjusted actually according to the needs of all programs, and the cleaning liquid storage area is fan-shaped (the embodiment and the attached drawings are fan-shaped;
a cleaning liquid overflow port (14) is formed in the cleaning liquid storage tank (2), and the cleaning liquid overflow port (14) is communicated with the cleaning liquid flow channel (15); the cleaning solution flow channel (15) is positioned on the outer wall of the cleaning solution storage tank and is communicated with the cracking cleaning tank (6);
a pyrolysis solution overflow port (12) is formed in the pyrolysis solution storage tank (3), and the pyrolysis solution overflow port (12) is communicated with the pyrolysis cleaning tank (6);
the eluent storage tank (4) is provided with an eluent overflow port (17), and the eluent overflow port (17) is communicated with the elution tank (7);
the oil storage tank (5) is provided with an amplification flow channel (18) facilitating oil to flow out, and the amplification flow channel (18) is communicated with the amplification tank (8).
As for the above cleaning liquid overflow port (14), the lysate overflow port (12), the eluent overflow port (17), and the amplification flow channel (18), the overflow ports are disposed on the arc-shaped sides of the corresponding storage tanks in the present embodiment and the drawings, so as to facilitate the liquid outflow.
The sample adding groove (1), the cleaning solution storage groove (2), the lysis solution storage groove (3), the eluent storage groove (4) and the oil storage groove (5) are grooves, and gaps are contained or not contained between adjacent grooves; the same cleaning solution storage tanks (2) may or may not contain a gap therebetween;
the plurality of amplification chambers (8) according to the present embodiment may or may not include gaps therebetween.
The working principle of the integrated diagnostic kit is described by combining the use method of the integrated diagnostic kit as follows:
a) the kit is placed on a matched workbench with a motor, a PCR sample is added into the sample adding slot (1), then the kit is placed into a detection instrument, and the kit and the motor are connected to form a whole. Adding a lysis solution into the lysis solution storage tank (3) (in the embodiment, the lysis solution can be packaged in a lysis solution bag in advance and placed in the lysis solution storage tank, opening the liquid bag to release the liquid in a puncturing or squeezing mode during use), driving the kit to rotate by a starting motor, enabling a sample and the lysis solution to respectively enter the lysis cleaning tank (6) from the sample adding flow channel (13) and the lysis solution overflow port (12) under the action of centrifugal force, and placing magnetic beads in PCR (polymerase chain reaction) in the lysis cleaning tank or the lysis solution in advance and also entering the same. At the moment, a sample, lysis solution and magnetic beads are arranged in the lysis cleaning tank (6), the magnetic beads are dispersed by adopting an oscillating and stirring mode, and the gene segments are adsorbed by the magnetic beads after the sample is fully lysed; the permanent magnet or the electromagnet is arranged below the cracking cleaning tank (6), after the gene fragments are fully adsorbed, the permanent magnet rises or the electromagnet is electrified to generate magnetic force on magnetic beads in the cracking cleaning tank (6), the magnetic beads are clustered and close to the bottom of the cracking cleaning tank (6), the motor rotates, and at the moment, the cracking liquid in the cracking cleaning tank (6) is conveyed to the waste liquid storage tank (9) through the cleaning waste liquid flow channel (10).
b) Cleaning solution is added into the cleaning solution storage tank (2) (in the embodiment, the cleaning solution can be packaged in a cleaning solution bag in advance and placed in the cleaning solution storage tank, when the device is used, the liquid bag is opened in a puncturing or squeezing mode to release liquid), the cleaning solution is conveyed into the cracking cleaning tank (6) from the cleaning solution overflow port (14) by the centrifugal force of the motor, the permanent magnet descends or the electromagnet is powered off, and the magnetic beads are dispersed in a mode that the motor drives the kit to vibrate and stir and are fully contacted with the cleaning solution to be cleaned; and the permanent magnet or the electromagnet is lifted again to be electrified, the magnetic beads are gathered and are close to the bottom, and the centrifugal force of the motor conveys the cleaning waste liquid to a waste liquid storage tank (9) through a cleaning waste liquid flow channel (10) to finish a cleaning process. The cleaning process can be repeated for a plurality of times according to the requirement so as to meet the cleaning requirement.
c) After the cleaning step is finished, magnetic beads are adsorbed and moved by magnetic force, the magnetic beads enter an elution tank (7) through a magnetic bead moving channel (16), eluent is added into an eluent storage tank (4) (in the embodiment, the eluent can be packaged in an eluent bag in advance and placed in the eluent storage tank, when the elution tank is used, a liquid bag is opened in a pricking or squeezing mode to release liquid), the eluent is driven by the centrifugal force of a motor to enter the elution tank (7) from an eluent overflow port (17), the magnetic beads are released by a magnet and are stirred in a vibrating mode, and the magnetic beads are dispersed and fully mixed with the eluent to be eluted; magnetic force is started again to gather the magnetic beads, the moving magnet returns the magnetic beads to the cracking cleaning tank (6) through the magnetic bead moving channel (16), at the moment, the motor is started, and eluent with gene segments enters the amplification tank (8) through the capillary flow channel (11).
d) Add oil in the oil holding tank (5), the motor is rotatory, and oil gets into amplification groove (8) from amplification runner (18) (in this embodiment, oil can pack in oil bag in advance and place the oil holding tank in, opens liquid package release oil through pricking during the use or extruded mode), and the motor lasts rotatoryly this moment, and amplification groove (8) are through the control of going up and down the temperature for the circulation that the PCR amplification system goes up and down the temperature. And detecting the fluorescent signal of the amplification tank after the temperature-changing circulation is finished to determine whether the target gene fragment exists in the sample. Wherein the number of amplification chambers (8) is set according to whether multiplex PCR is required, freeze-dried reagent components required for PCR amplification are pre-stored in the plurality of amplification chambers (8), and the freeze-dried reagent components comprise primers required for amplification reaction and other components required for PCR amplification, and the primers in different amplification chambers (8) are different.
It should be noted that, in this document, the term "comprises/comprising" or any other variation thereof is intended to cover a non-exclusive inclusion, so that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but also other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. An integrated diagnostic kit is characterized by comprising a disc-shaped kit body (19), and a sample loading slot (1), a cleaning solution storage slot (2), a lysis solution storage slot (3), an eluent storage slot (4), an oil storage slot (5), a lysis cleaning slot (6), an elution slot (7), an amplification slot (8) and a waste liquid storage slot (9) which are distributed on the kit body (19);
the sample adding slot (1) is positioned in the middle of the kit; the cleaning solution storage tank (2), the lysis solution storage tank (3), the eluent storage tank (4) and the oil storage tank (5) are concentrically arranged with the sample loading slot (1); the position of the amplification tank (8) is close to the edge of the kit, and the cracking cleaning tank (6) and the elution tank (7) are close to the liquid storage area;
the sample loading groove (1) is communicated with the cracking cleaning groove (6) through a loading flow channel (13); the cracking cleaning tank (6) is communicated with the waste liquid storage tank (9) through a cleaning waste liquid flow channel (10); the lysis cleaning tank (6) is communicated with the elution tank (7) through a magnetic bead moving channel (16); the elution tank (7) is communicated with the amplification tank (8) through a capillary flow channel (11);
at least one of the cleaning solution storage tank (2), the lysis solution storage tank (3), the eluent storage tank (4) and the oil storage tank (5) is arranged;
a cleaning liquid overflow port (14) is formed in the cleaning liquid storage tank (2), and the cleaning liquid overflow port (14) is communicated with a cleaning liquid flow channel (15); the cleaning solution flow passage (15) is communicated with the cracking cleaning tank (6);
a pyrolysis solution overflow port (12) is formed in the pyrolysis solution storage tank (3), and the pyrolysis solution overflow port (12) is communicated with the pyrolysis cleaning tank (6);
the eluent storage tank (4) is provided with an eluent overflow port (17), and the eluent overflow port (17) is communicated with the elution tank (7);
the oil storage tank (5) is provided with an amplification flow channel (18) facilitating oil to flow out, and the amplification flow channel (18) is communicated with the amplification tank (8).
2. The integrated amplification kit according to claim 1, wherein the sample addition slot (1) has a circular shape and is located at the center of the kit body (19).
3. The integrated amplification kit according to claim 1, wherein the cleaning solution storage tank (2), the lysis solution storage tank (3), the eluent storage tank (4) and the oil storage tank (5) are arranged around the sample loading tank (1) and are all in a fan shape.
4. The integrated amplification kit according to claim 1, wherein the amplification chamber (8) comprises two or more chambers, and the chambers are sequentially connected and arranged concentrically with the sample addition chamber (1).
5. The integrated amplification kit according to claim 1, wherein the number of the cleaning solution storage tanks (2) is two or more, each cleaning solution storage tank (2) is provided with an overflow port (14), the overflow ports (14) are respectively communicated with a cleaning solution flow channel (15), and the cleaning solution flow channel is annular and is located on the outer wall of the cleaning solution storage tank (2).
6. The integrated diagnostic kit according to claim 1, wherein the waste reservoir (9) is arranged concentrically with the sample application well (1) and close to the edge of the kit.
7. The integrated diagnostic kit according to claim 1, wherein the washing waste liquid flow channel (10) is U-shaped or S-shaped; the capillary flow channel (11) is U-shaped or S-shaped.
8. A method for detecting a target gene using the integrated diagnostic kit of claim 1, comprising the steps of:
s1, placing a kit on a matched workbench with a motor, adding a PCR sample into a sample adding groove (1), adding a lysis solution into a lysis solution storage groove (3), starting the motor, synchronously rotating the kit, allowing the sample and the lysis solution to enter a lysis cleaning groove (6) under the action of centrifugal force, adding PCR magnetic beads, and performing lysis reaction on the sample; after the reaction, magnetic force is added, and the centrifugal force of a motor centrifugally flows the reacted lysate into a waste liquid storage tank (9);
s2, removing the magnetic force, adding cleaning solution into the cleaning solution storage tank (2), starting a motor, stopping the motor after the cleaning solution flows into the cracking cleaning tank (6), and cleaning under vibration and stirring; after the washing is finished, magnetic force is applied, and the gathered magnetic beads enter the elution tank (7) along the magnetic bead channel (16); starting a motor, and centrifugally flowing the cleaning waste liquid into a waste liquid storage tank (9);
s3, adding eluent into the eluent storage tank (4), rotating with a motor to enter an elution tank, removing magnetic force, stopping the motor, carrying out vibration stirring elution operation, then adding magnetic force to remove magnetic beads, starting the motor, and enabling the eluent to enter an amplification tank (8) through a capillary flow channel (11);
s4, adding oil into the oil storage tank (5), rotating with the motor to enter an amplification tank (8), controlling the temperature-variable circulation of a cavity of the amplification tank to carry out amplification reaction, and detecting a fluorescence signal of the amplification tank after the reaction is finished to obtain a detection result.
9. The method for detecting a target gene according to claim 6, wherein the step S2 is repeated two or more times.
10. The method of claim 6, wherein the amplification chamber (8) of step S4 contains lyophilized reagents required for PCR amplification, the lyophilized reagents comprising primers required for amplification and other components required for PCR amplification.
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| CN202010826506.3A CN111925925B (en) | 2020-08-17 | 2020-08-17 | Integrated diagnostic kit and application thereof |
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| CN202010826506.3A CN111925925B (en) | 2020-08-17 | 2020-08-17 | Integrated diagnostic kit and application thereof |
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|---|---|---|---|---|
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