CN219032123U

CN219032123U - Nucleic acid purification and detection reaction liquid preparation device

Info

Publication number: CN219032123U
Application number: CN202223003561.6U
Authority: CN
Inventors: 喻学锋; 周文华; 舒伟良; 惠允
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2022-11-10
Filing date: 2022-11-10
Publication date: 2023-05-16
Anticipated expiration: 2032-11-10

Abstract

The utility model relates to a nucleic acid purification and detection reaction solution preparation device, which comprises an upper cover layer and a reagent storage layer rotatably arranged below the upper cover layer, wherein the upper cover layer comprises an upper cover main body, a sample sealing cover and a silica gel sleeve which are arranged on the upper cover main body at intervals; the reagent storage layer comprises a reagent storage main body and a plurality of liquid pools which are arranged on the reagent storage layer at intervals and used for storing processing liquid for processing sample nucleic acid; the device can realize the whole flow operation of sample pyrolysis, nucleic acid adsorption, nucleic acid cleaning, nucleic acid elution and detection reaction liquid under the totally enclosed environment through the design of rotatable main body and the combination of the silica gel sleeve and the magnetic sleeve extraction method, and the device has simple integral structure and simple operation, and can be compatible with various types of samples.

Description

Nucleic acid purification and detection reaction liquid preparation device

Technical Field

The utility model relates to the technical field of biomedicine, in particular to a device for preparing a nucleic acid purification and detection reaction solution.

Background

Early, rapid and accurate disease detection is critical to maximize crisis management efficiency, therapeutic efficacy, and economic stability. However, current testing practices are primarily limited to centralized laboratories, where typically patient samples are taken to a hospital or clinic for testing, the results of which are fed back over several days, and disease testing is often longer in developing countries or regions due to lack of skilled personnel and medical infrastructure. Thus, the need for portable, easy-to-use, and point-of-care testing (POCT) is rapidly increasing.

Molecular diagnostics is revolutionizing the clinical detection of infectious diseases, one significant advancement is the reduction in the time required to diagnose infectious diseases. Polymerase Chain Reaction (PCR) is the most commonly used molecular diagnostic technique, but thermal cycling requires an energy-consuming instrument, and various isothermal amplification reactions such as loop-mediated isothermal amplification (LAMP), rolling Circle Amplification (RCA) and Recombinant Polymerase Amplification (RPA) have been developed in order to simplify and shorten the time of thermal cycling, and on the basis of these techniques, many commercial kits have been developed to achieve highly sensitive and rapid detection of pathogens. However, the relatively large number of steps in sample preparation and nucleic acid extraction of these kits has limited their wide application in clinical diagnosis. Therefore, it has become a trend to develop a lab-on-a-chip system that integrates all the steps of molecular diagnostics into one miniaturized device. In POCT diagnosis, a patient can collect samples by himself without the help of medical staff, the collected samples can be immediately analyzed and disease screened at a sampling point, and the detection of the biomarker can be realized only by a very small sample size.

In the prior art, the centrifugal column method or the magnetic bead method is generally used for extracting nucleic acid, and steps such as cracking, combining, eluting and the like are generally needed, so that the whole full-automatic instrument for 'sample inlet and outlet' is very difficult to realize. In terms of transfer of the effective components in each step, a manual transfer mode is adopted in the prior art, so that the operation is complex, time and labor are wasted, samples are difficult to sufficiently transfer, the efficient transfer is realized, the result is unstable easily caused by manual operation, and the detection implementation difficulty is high. In addition, the prior art basically adopts complicated liquid way valves to transfer liquids with different functions, so that the use cost of consumable materials can be increased for large-scale application, and the operation difficulty and instability can be greatly increased for the control of the technology. In addition, the main flow technology of molecular detection is a fluorescent quantitative PCR technology, and the PCR technology has the characteristic of exponentially amplifying templates, so that the whole operation process is easy to cause PCR aerosol pollution due to the existing open consumable materials. For example, chinese patent application publication No. CN101452003a discloses a liquid pool micropump fully integrated in a microfluidic chip, where the liquid pool micropump is composed of a closed filtering liquid pool, a pumping liquid pool and a microchannel integrated on the microfluidic chip, and only a part of functions of nucleic acid detection can be implemented, and practical requirements of clinical users are not considered, which is difficult to be put into practical use.

Disclosure of Invention

The utility model aims to provide a nucleic acid purification and detection reaction liquid preparation device which can realize the full-flow operation of sample cracking, nucleic acid adsorption, nucleic acid cleaning, nucleic acid elution and detection reaction liquid in a fully-closed environment, has a simple integral structure and is easy to operate, and can be compatible with various types of samples.

In one aspect, the present utility model provides a nucleic acid purification and detection reaction solution preparation device, comprising:

the upper cover layer comprises an upper cover main body, a sample sealing cover and a silica gel sleeve, wherein the sample sealing cover and the silica gel sleeve are arranged on the upper cover main body at intervals; and

the reagent storage layer is rotatably arranged below the upper cover layer and comprises a reagent storage main body and a plurality of liquid pools which are arranged on the reagent storage layer at intervals, wherein the liquid pools are used for storing treatment liquid for treating sample nucleic acid;

wherein the sample sealing cap is used to deliver a sample to the liquid reservoir of the reagent storage layer and maintain the tightness of the reagent storage layer; the silica gel sleeve is of an elastic telescopic tubular structure and is used for being vertically telescopic to be matched with the inserted magnetic rod and the magnetic rod sleeve to realize the attraction and transfer of the magnetic beads adsorbed with nucleic acid in the corresponding liquid pool, so that the full-closed nucleic acid purification treatment and the preparation of nucleic acid detection reaction liquid are realized.

In one embodiment of the present utility model, the reagent storage layer and the upper cover layer are rotatably connected by adopting a transmission latch structure; the range of each rotation angle of the reagent storage layer is 15-90 degrees.

In an embodiment of the utility model, the upper cover layer further comprises a convex part with a central groove extending from the upper cover main body, and the silica gel sleeve is installed in the central groove of the convex part; the reagent storage main body is provided with a cylindrical structure and a columnar structure extending from the cylindrical structure, the diameter of the columnar structure is smaller than that of the cylindrical structure, and the columnar structure corresponds to the liquid pool one by one.

In one embodiment of the present utility model, the plurality of liquid reservoirs include a magnetic bead storage liquid reservoir for storing magnetic beads that adsorb nucleic acids, a lysis liquid reservoir for storing a sample lysis liquid, a washing liquid reservoir for storing a nucleic acid washing liquid, and an eluent and a detection reagent storage liquid reservoir for storing a nucleic acid eluent and a detection reagent freeze-dried ball wrapped with paraffin.

In an embodiment of the utility model, the three cleaning solution tanks include a first cleaning solution tank, a second cleaning solution tank and a third cleaning solution tank which are sequentially arranged.

In an embodiment of the utility model, the magnetic bead preserving solution tank, the lysing solution tank, the first washing solution tank, the second washing solution tank, the third washing solution tank, the eluent and the detection reagent preserving solution tank are sequentially and uniformly arranged at intervals along the circumferential direction of the reagent storage layer.

In an embodiment of the utility model, the silica gel sleeve is of an elastic telescopic tubular structure, and a sealing silica gel pad is arranged between the upper cover layer and the reagent storage layer.

In an embodiment of the utility model, the upper cover main body and the reagent storage layer are both prepared by injection molding.

In one embodiment of the utility model, the sample is sputum, tissue, blood or stool; the detection reagent in the detection reagent freeze-dried ball is a PCR (polymerase chain reaction), RPA (reverse transcription amplification) or LAMP (loop-mediated isothermal amplification) reagent; the whole length of the nucleic acid purification and detection reaction liquid preparation device is 35mm, the width is 35mm, and the height is 43mm.

The utility model also provides a preparation method of the nucleic acid purification and detection reaction liquid, which comprises the following steps executed by adopting the preparation device of the nucleic acid purification and detection reaction liquid:

s1, opening a sample sealing cover to deliver a sample in a cracking liquid pool so as to realize sample cracking;

s2, inserting a magnetic rod and a magnetic rod sleeve into the silica gel sleeve of the upper cover layer, and attracting the magnetic beads in the magnetic bead preservation solution to the silica gel sleeve through the magnetic rod;

s3, rotating the reagent storage layer according to a preset angle in sequence, enabling the silica gel sleeve to be aligned with a corresponding liquid pool of the reagent storage layer in sequence, and when the silica gel sleeve is aligned with the corresponding liquid pool each time, releasing magnetic beads in the corresponding liquid pool by means of pulling a magnetic rod out of the magnetic rod sleeve, then attracting the magnetic beads again by means of inserting the magnetic rod and driving the silica gel sleeve to extend into the corresponding liquid pool, and then sequentially transferring the magnetic beads in the corresponding liquid pool by repeating releasing and attracting operations of the magnetic beads, so that the processes of adsorbing sample nucleic acid, cleaning nucleic acid, eluting nucleic acid and preparing nucleic acid detection reaction liquid by the magnetic beads are sequentially achieved.

In one embodiment of the utility model, the reagent storage layer is set at an angle of 15 to 90 ° per rotation.

In an embodiment of the present utility model, the step S2 specifically includes the steps of:

s21, inserting the magnetic rod and the magnetic rod sleeve into the silica gel sleeve of the upper cover layer, driving the silica gel sleeve to stretch downwards into the magnetic bead preservation liquid pool through the magnetic rod and the magnetic rod sleeve, attracting the magnetic beads to the top of the silica gel sleeve, and after attraction is completed, moving the magnetic rod and the magnetic rod sleeve upwards to an initial position state of the silica gel sleeve.

In an embodiment of the utility model, the reagent storage layer further includes a lysis solution tank for storing a sample lysis solution, a washing solution tank for storing a nucleic acid washing solution, and an eluent and a detection reagent preservation solution tank for storing a nucleic acid eluent and a detection reagent freeze-dried ball wrapped by paraffin, wherein the washing solution tank includes a first washing solution tank, a second washing solution tank and a third washing solution tank which are sequentially arranged; the step S3 comprises the steps of:

s31, rotating the reagent storage layer by 60 degrees to enable the silica gel sleeve to be located right above the cracking liquid pool, driving the magnetic rod and the magnetic rod sleeve to enable the silica gel sleeve to stretch downwards and stretch into the cracking liquid pool, and enabling the magnetic rod to be pulled away from the magnetic rod sleeve, so that a magnetic field disappears, and enabling magnetic beads adsorbed by the silica gel sleeve to be released into the cracking liquid pool, and adsorbing nucleic acid in the cracking liquid pool through the magnetic beads;

s32, inserting the magnetic rod into the magnetic rod sleeve again and driving the silica gel sleeve to extend into the cracking liquid pool, attracting the magnetic beads adsorbed with nucleic acid in the cracking liquid pool to the top of the silica gel sleeve, and after attraction is completed, moving the magnetic rod and the magnetic rod sleeve upwards to an initial position state of the silica gel sleeve;

s33, rotating the reagent storage layer by 60 degrees, so that the silica gel sleeve inserted with the magnetic rod and the magnetic rod sleeve is positioned right above the first cleaning liquid pool, driving the magnetic rod and the magnetic rod sleeve to downwards stretch and retract into the first cleaning liquid pool, and pumping the magnetic rod away from the magnetic rod sleeve, so that a magnetic field disappears, and enabling magnetic beads adsorbed by the silica gel sleeve to enter the cleaning liquid pool, thereby realizing nucleic acid cleaning;

s34, inserting the magnetic rod into the magnetic rod sleeve again and driving the silica gel sleeve to extend into the first cleaning liquid pool, attracting the cleaned magnetic beads to the top of the silica gel sleeve, and after attraction is completed, moving the magnetic rod and the magnetic rod sleeve upwards to an initial position state of the silica gel sleeve;

s35, completing the cleaning process of the magnetic beads in the second cleaning liquid pool and the third cleaning liquid pool according to the same operation as that of the steps S33 and S34;

s36, rotating the reagent storage layer by 60 degrees to enable the silica gel sleeve to be located right above the eluent and detection reagent storage liquid pool, driving the silica gel sleeve to stretch downwards through the magnetic rod and the magnetic rod sleeve to enter the eluent and detection reagent storage liquid pool, and pumping the magnetic rod away from the magnetic rod sleeve to enable the magnetic field to disappear so that magnetic beads adsorbed by the silica gel sleeve enter the eluent and detection reagent storage liquid pool, thereby realizing nucleic acid elution;

s37, inserting the magnetic rod into the magnetic rod sleeve again, driving the silica gel sleeve to extend into the eluent and detection reagent preservation liquid pool, attracting the magnetic beads eluted with nucleic acid to the top of the silica gel sleeve, and after attraction is completed, moving the magnetic rod and the magnetic rod sleeve upwards to an initial position state of the silica gel sleeve;

s38, heating the eluent and the detection reagent preservation liquid pool to dissolve paraffin which wraps the detection reagent freeze-dried ball in the eluent and the detection reagent preservation liquid pool, floating the dissolved paraffin to the uppermost layer of the eluent and the detection reagent preservation liquid pool, and dissolving the detection reagent freeze-dried ball in the nucleic acid eluent to treat nucleic acid, thereby completing preparation of the detection reaction liquid.

In an embodiment of the present utility model, the step S1 further includes the steps of: and when the sample is cracked, heating and ultrasonic treatment are further carried out on the cracking liquid pool, and the sample cracking time is 1-5 min.

In one embodiment of the present utility model, in step S31, the time for the nucleic acid adsorption process is 1 to 10 minutes.

In an embodiment of the present utility model, the washing time of the magnetic beads in the first washing liquid tank, the second washing liquid tank and the third washing liquid tank is 1-2 min, and the magnetic beads are dried for 1-5 min after being washed in the first washing liquid tank, the second washing liquid tank and the third washing liquid tank.

In one embodiment of the present utility model, in step S36, the time for the nucleic acid eluting process is 1 to 5 minutes.

The utility model has the following beneficial effects:

(1) The nucleic acid purification and detection reaction liquid preparation device can realize the full-flow operation of sample cracking, nucleic acid adsorption, nucleic acid cleaning, nucleic acid elution and detection reaction liquid in a fully-closed environment, is beneficial to saving the pretreatment time of nucleic acid detection and improves the nucleic acid detection efficiency.

(2) The utility model realizes the closed environment of a plurality of liquid pools by combining the upper cover layer and the reagent storage layer, thereby ensuring that sample cracking, nucleic acid combination, cleaning, elution and preparation of detection reaction liquid are realized in a completely closed state, ensuring the tightness of the nucleic acid extraction and expansion process, reducing the interference of external aerosol on detection results and preventing the infection hazard of the sample to detection personnel.

(3) The reagent storage layer comprises a plurality of liquid pools, the treatment liquid types of the liquid pools can be set according to the nucleic acid purification and reaction liquid preparation requirements of different samples, and the reagent storage layer can be compatible with the nucleic acid purification and detection reaction liquid preparation requirements of various samples.

(4) The utility model can reduce the arrangement of the flow path switch control valve through the design of the rotary main body, realizes the transfer of magnetic beads in different liquid storage cavities through rotating the reagent storage main body structure, and provides a sample nucleic acid purification and detection reaction liquid preparation device with simple structure and simple operation.

(5) The nucleic acid purification and detection reaction solution preparation device provided by the utility model has the advantages that the eluent and the detection reagent freeze-dried balls are placed in one liquid pool, so that the operation of transferring the nucleic acid eluent in the preparation process of the detection reaction solution is solved, the liquid path of the device can be omitted, and the operation of liquid control is avoided.

(6) The utility model adopts the freeze-dried ball to store the detection reagent, and wraps a layer of paraffin on the outer surface of the freeze-dried ball of the detection reagent, thereby ensuring that the freeze-dried ball of the detection reagent and the nucleic acid eluent can be placed in a liquid pool, and solving the coexistence problem of the detection reagent and the nucleic acid eluent.

(7) The preparation device for the nucleic acid purification and detection reaction liquid adopts an injection molding mode to realize preparation, can realize batch production, and has the advantages of small batch-to-batch difference, high yield and low cost.

(8) The nucleic acid purification and detection reaction solution preparation device can be combined with the existing PCR, LAMP, RCA and RPA external matched instruments, and is beneficial to realizing the full-automatic detection process of sample inlet and outlet.

Further objects and advantages of the present utility model will become fully apparent from the following description and the accompanying drawings.

Drawings

FIG. 1 is a schematic perspective view of a device for preparing a nucleic acid purification and detection reaction solution according to a preferred embodiment of the present utility model;

FIG. 2 is an exploded view of the nucleic acid purification and detection reaction solution preparing apparatus shown in FIG. 1;

FIG. 3 is a schematic plan view of the nucleic acid purification and detection reaction solution preparation apparatus shown in FIG. 1;

FIG. 4 is a schematic cross-sectional view of the nucleic acid purification and detection reaction solution preparation apparatus shown in FIG. 3 along the A-A axis;

FIG. 5 is a schematic plan view of the reagent reservoir layer of the nucleic acid purification and detection reaction solution preparation apparatus shown in FIG. 1.

Reference numerals illustrate: a nucleic acid purification and detection reaction solution preparation device 100; an upper cover layer 10; an upper cover main body 11; a sample sealing cap 12; a silica gel cover 13; a convex portion 14; a central slot 14; a reagent storage layer 20; a reagent storage main body 21; a cylindrical structure 211; columnar structures 212; a liquid bath 22; a magnetic bead holding liquid pool 221; a lysate tank 222; a cleaning liquid bath 223; a first cleaning liquid bath 2231; a second cleaning liquid bath 2232; a third cleaning liquid bath 2233; eluent and detection reagent reservoir 224.

Detailed Description

The following description is presented to enable one of ordinary skill in the art to make and use the utility model. The preferred embodiments in the following description are by way of example only and other obvious variations will occur to those skilled in the art. The basic principles of the utility model defined in the following description may be applied to other embodiments, variations, modifications, equivalents, and other technical solutions without departing from the spirit and scope of the utility model.

It will be appreciated by those skilled in the art that in the present disclosure, the terms "vertical," "transverse," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," etc. refer to an orientation or positional relationship based on that shown in the drawings, which is merely for convenience of description and to simplify the description, and do not indicate or imply that the apparatus or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore the above terms should not be construed as limiting the present utility model.

It will be understood that the terms "a" and "an" should be interpreted as referring to "at least one" or "one or more," i.e., in one embodiment, the number of elements may be one, while in another embodiment, the number of elements may be plural, and the term "a" should not be interpreted as limiting the number.

In the description of the present utility model, it should be noted that, unless explicitly specified and limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be either fixedly connected, detachably connected, or integrally connected, for example; can be mechanically connected, electrically connected or can be communicated with each other; can be directly connected or indirectly connected through an intermediate medium, and can be communicated with the inside of two elements or the interaction relationship of the two elements. The specific meaning of the above terms in the present utility model can be understood by those of ordinary skill in the art according to the specific circumstances.

As shown in fig. 1 to 5, a specific structure of a nucleic acid purification and detection reaction solution preparation apparatus 100 according to a preferred embodiment of the present utility model is illustrated. The nucleic acid purification and detection reaction solution preparation device 100 is used for extracting and purifying sample nucleic acid and preparing detection reaction solution, and can be combined with an external matched instrument to realize the whole flow operation of sample processing and detection. The external matched instrument can be PCR, LAMP, RCA, RPA and other instruments, and the utility model is not limited to this. Correspondingly, the detection reaction liquid prepared by the device is PCR, LAMP, RCA and RPA detection reaction liquid, and the utility model is not limited to this.

The nucleic acid purification and detection reaction solution preparation device 100 can realize the whole-flow operation of sample cracking, nucleic acid adsorption, nucleic acid cleaning, nucleic acid elution and detection reaction solution in a totally enclosed environment, is beneficial to saving the pretreatment time of detection and improves the detection efficiency.

Specifically, the nucleic acid purification and detection reaction solution preparation device 100 is composed of two layers of structures, namely an upper cover layer 10 and a reagent storage layer 20 rotatably arranged below the upper cover layer 10, and can realize sample treatment and detection reaction solution preparation in a totally enclosed environment through the cooperation of the upper cover layer 10 and the reagent storage layer 20.

It can be appreciated that the utility model can reduce the arrangement of the flow path switch control valve by the design of the rotary reagent storage layer, and realize the transfer of magnetic beads in different liquid storage cavities by rotating the reagent storage layer 20, thereby realizing the different treatments of nucleic acid, and providing the nucleic acid purification and detection reaction solution preparation device 100 of a sample with simple structure and simple operation.

More specifically, the reagent storage layer 20 includes a reagent storage main body 21 and a plurality of liquid reservoirs 22 provided at intervals on the reagent storage layer 20, and in this preferred embodiment, the plurality of liquid reservoirs 22 include a magnetic bead storage liquid reservoir 221 for storing magnetic beads adsorbing nucleic acids, a lysate liquid reservoir 222 for storing a sample lysate, a washing liquid reservoir 223 for storing a nucleic acid washing liquid, and an eluent and detection reagent storage liquid reservoir 224 for storing nucleic acid eluent and detection reagent freeze-dried balls wrapped with paraffin.

It should be appreciated that in some embodiments of the present utility model, the fluid reservoir 22 may be a fluid reservoir containing other types of processing fluids, and may be adapted to actual sample processing and testing requirements, as the present utility model is not limited in this regard.

It can be appreciated that the nucleic acid purification and detection reaction solution preparation device 100 of the present utility model places the eluent and the detection reagent freeze-dried balls in one liquid pool, thereby solving the problem that the nucleic acid eluent needs to be transferred in the preparation process of the detection reaction solution, omitting the liquid path of the device and avoiding the operation of liquid control.

In addition, the utility model adopts the freeze-dried ball to store the detection reagent, and wraps a layer of paraffin on the outer surface of the freeze-dried ball of the detection reagent, thereby avoiding the freeze-dried ball of the detection reagent from being dissolved in the nucleic acid eluent, ensuring that the freeze-dried ball of the detection reagent and the nucleic acid eluent can be placed in a liquid pool, and solving the coexistence problem of the detection reagent and the nucleic acid eluent.

In addition, the paraffin also has the function of solving the problem of sample evaporation in the process of PCR heating and preventing PCR aerosol pollution, and the paraffin wrapped outside the freeze-dried balls can be dissolved and float on the solution by heating the eluent and the detection reagent preservation liquid pool 224 in the process of preparing the detection reaction liquid, so that the sealing function of the solution below is achieved, and the sample evaporation and aerosol pollution after heating are prevented.

It should be noted that the content of the freeze-dried pellet can be changed according to the need, including the reagent system and the probe system used in RPA, LAMP and other PCR methods, and the utility model is not limited thereto.

Specifically, the upper cover layer 10 includes an upper cover main body 11, and a sample sealing cover 12 and a silica gel cover 13 which are disposed at intervals on the upper cover main body 11, wherein the sample sealing cover 12 is used for delivering a sample to the lysis solution pool 222 of the reagent storage layer 20 and maintaining the sealability of the reagent storage layer 20; the silica gel sleeve 13 is used for inserting the magnetic rod and the magnetic rod sleeve, and is used for stretching up and down to be matched with the magnetic rod and the magnetic rod sleeve to realize attraction and transfer of the magnetic beads in the corresponding liquid pools.

It should be noted that the reagent storage layer 20 and the upper cover layer 10 may be rotatably connected by a driving latch structure; the reagent storage layer 20 is also provided with different rotation angles according to the number of the liquid pools 22 of the reagent storage layer 20, and each rotation angle of the reagent storage layer 20 ranges from 15 to 90 °.

In particular, the silica gel sleeve 13 is of an elastic telescopic tubular structure, so that the silica gel sleeve 13 can be stretched up and down, after the magnetic rod and the magnetic rod sleeve of an external matched instrument are inserted into the silica gel sleeve 13, the silica gel sleeve 13 can be carried to move down continuously and go deep into the liquid pool of the reagent storage layer 20, when different treatment liquids need to be replaced, the magnetic rod and the magnetic rod sleeve move up to the initial position of the silica gel sleeve 13, the reagent storage layer 20 is rotated, and the liquid pool to be reacted next is rotated to the position right below the silica gel sleeve 13. After the rotation is completed, the magnetic rod and the magnetic rod sleeve can drive the silica gel sleeve 13 to move downwards to the corresponding treatment liquid for reaction. When the magnetic beads are required to be released into the treatment liquid, the magnetic rod and the magnetic rod sleeve are separated, the magnetic rod moves upwards, the magnetic rod sleeve is kept still, and the magnetic beads adsorbed outside the silica gel sleeve 13 are dispersed into the liquid due to the disappearance of the magnetic field so as to perform more sufficient reaction.

That is, the nucleic acid purification and detection reaction solution preparing apparatus 100 performs sample lysis by opening the sample sealing cap 12 to deliver a sample in the lysis solution tank 222, aligns the silica gel cover 13 with a corresponding solution tank by rotating the reagent storage layer 20, further performs adsorption of nucleic acid by combining up-and-down expansion and magnetic rod extraction of the silica gel cover 13, and performs treatment and transfer of nucleic acid-adsorbed magnetic beads in a corresponding solution tank, thereby performing purification treatment of nucleic acid of a sample and detection reaction solution preparation in a totally enclosed manner.

It should be noted that the upper cover 10 further includes a protrusion 14 extending from the upper cover body 11 and having a central slot 14; that is, the protrusion 14 has a hollow cylindrical structure for defining an installation space for forming the silicone jacket 13, that is, the silicone jacket 13 is installed in the central groove 14 of the protrusion 14.

It should be further noted that in a preferred embodiment of the present utility model, the number of the cleaning solution tanks 223 may be three, including a first cleaning solution tank 2231, a second cleaning solution tank 2232, and a third cleaning solution tank 2233, which are sequentially disposed; the magnetic bead storage liquid tanks 221, the lysing liquid tank 222, the first cleaning liquid tank 2231, the second cleaning liquid tank 2232, the third cleaning liquid tank 2233, and the eluting solution and detection reagent storage liquid tanks 224 are sequentially and uniformly arranged at intervals along the circumferential direction of the reagent storage layer 20, that is, in this preferred embodiment, six liquid tanks are arranged on the reagent storage layer 20 at intervals, so that the reagent storage layer 20 can align the silica gel cover 13 with the corresponding liquid tanks by rotating 60 ° each time.

In some embodiments of the present utility model, the number of the cleaning solution tanks 223 may be other, such as four, five or more, which is not limited in this aspect of the present utility model, and may be set according to actual detection requirements.

That is, the number of the liquid pools provided on the reagent storage layer 20 may be set according to actual requirements, and the type of the treatment liquid in the liquid pools may be set according to the nucleic acid purification and reaction liquid preparation requirements of different samples, so that the nucleic acid purification and detection reaction liquid preparation device 100 of the present utility model may be compatible with the nucleic acid purification and detection reaction liquid preparation requirements of samples in various forms. Optionally, the sample is any one of sputum, tissue, blood or stool.

It should be noted that a sealing silica gel pad is further provided between the upper cover 10 and the reagent storage layer 20 to further ensure the sealing performance of the nucleic acid purification and detection reaction solution preparation apparatus 100.

It should be noted that the upper cover 10 and the reagent storage layer 20 further have corresponding central through holes, and the liquid storage body has a two-part structure, i.e. an upper half is a cylindrical structure with an integral structure, a lower half is a cylindrical structure with a smaller diameter formed by extending the cylindrical structure 211 downward, the number of the cylindrical structures 212 corresponds to the number of the liquid pools 22, and the positions of the cylindrical structures 212 are in one-to-one correspondence, and the cylindrical structures 212 may be cylindrical or conical.

It can be understood that the present utility model realizes a closed environment of a plurality of liquid pools by combining the upper cover layer 10 and the reagent storage layer 20, and can ensure sample lysis, nucleic acid combination, washing, elution and preparation of detection reaction solution in a completely closed state by the rotatable design and combined magnetic sleeve extraction method of the reagent storage layer 20, thereby ensuring tightness of nucleic acid extraction and expansion processes, reducing interference of external aerosol on detection results, and preventing sample leakage from causing infection hazard to detection personnel.

It should be noted that, in this preferred embodiment of the present utility model, both the upper cover body 11 and the reagent storage layer 20 are manufactured by injection molding. The nucleic acid purification and detection reaction solution preparation apparatus 100 has an overall length of 35mm, a width of 35mm, and a height of 43mm, and may be provided in other dimensions as required, and the present utility model is not limited thereto.

The method of using the nucleic acid purification and detection reaction solution preparation apparatus 100 of the present utility model will be specifically described below, taking as an example the nucleic acid purification and detection reaction solution preparation apparatus 100 of the present utility model in combination with a PCR instrument to perform nucleic acid detection of a sample.

Sample pretreatment and PCR amplification detection:

1. sample pretreatment

A) Lysing the samples:

the collected samples comprise sputum, tissues, blood, feces and the like, the pretreatment of the samples is complex, and various treatment modes such as chemical lysate guanidine isothiocyanate, biological enzyme lysate proteinase K and heating and mechanical shearing are required to be compatible, so that different modes are configured corresponding to different objects.

After the sample is added into the cracking liquid pool 222 through the sample sealing cover 12, the sample sealing cover 12 is covered, the cracking of the sample is realized under the action of the cracking liquid in the cracking liquid pool 222, the outside of the liquid pool can be heated to 70 ℃ in the cracking process, and ultrasound is applied in the reaction process, so that the cracking of the sample is further promoted, and the process waits for 1-5 min.

B) Nucleic acid binding:

after the sample is cracked, the magnetic rod and the magnetic rod sleeve move downwards, extend into the silica gel sleeve 13 and drive the silica gel sleeve 13 to slowly enter the magnetic bead storage liquid pool 221, and attract the magnetic beads in the magnetic bead storage liquid pool 221 to the top of the silica gel sleeve 13. After the attraction of the magnetic beads is completed, the magnetic rod and the magnetic rod sleeve move upwards to the initial state position of the silica gel sleeve 13, and the reagent storage layer 20 is rotated clockwise by 60 degrees through the transmission of an external matching device (namely a PCR instrument), so that the cracking liquid pool 222 is positioned at the position right below the silica gel sleeve 13. The magnetic rod and the magnetic rod sleeve move downwards with the silica gel sleeve 13, enter into the pyrolysis liquid of the pyrolysis liquid pool 222, move upwards to leave the magnetic rod sleeve, namely, the magnetic rod is pulled out of the magnetic rod sleeve, and as the magnetic field disappears, the magnetic beads outside the silica gel sleeve 13 can be fully dispersed into the pyrolysis liquid under the vibration of the magnetic rod sleeve, and the adsorption of nucleic acid is realized in the mixing process, and the process waits for 1-10 min.

Nucleic acid cleaning:

after the magnetic beads are used for adsorbing nucleic acid, the magnetic rod and the magnetic rod sleeve carry the silica gel sleeve 13 to slowly move into the cracking liquid together, and the magnetic beads in the cracking liquid are adsorbed to the top position of the silica gel sleeve 13. And then moves upwards to the initial state position of the silica gel cover 13, and the reagent storage layer 20 is rotated clockwise by 60 degrees through the transmission of an external matching device, so that the first cleaning liquid pool 2231 is positioned right below the silica gel cover 13. The magnetic rod and the magnetic rod sleeve move downwards with the silica gel sleeve 13, enter the first cleaning solution, move upwards to leave the magnetic rod sleeve, and due to the fact that the magnetic field disappears, magnetic beads outside the silica gel sleeve 13 can be fully dispersed into the first cleaning solution under the vibration of the magnetic rod sleeve, wait for 1-2 min, continue to operate, adsorb the magnetic beads to the top of the silica gel sleeve 13, rotate the reagent storage layer 20, perform cleaning operation of the magnetic beads in the second cleaning solution pool 2232 and the third cleaning solution pool 2233, adsorb the magnetic beads to the top of the silica gel sleeve 13 after the cleaning of nucleic acid is completed, and dry for 1-5 min after moving to the initial state position of the silica gel sleeve 13.

C) Nucleic acid elution:

after the magnetic beads are dried, the reagent storage layer 20 is rotated clockwise by 60 degrees through the transmission of an external matching device, so that the eluent and the detection reagent storage liquid tank 224 are positioned right below the silica gel sleeve 13, the magnetic rod sleeve moves downwards into the eluent with the silica gel sleeve 13, the magnetic beads are fully dispersed into the eluent by vibrating the magnetic rod sleeve, and nucleic acid adsorbed by the magnetic beads is eluted through the eluent, so that a nucleic acid purification liquid is obtained; after waiting for 1-5 min, the magnetic rod is downward, and after the magnetic attraction is completed, the magnetic rod and the magnetic rod sleeve move upward with the silica gel sleeve 13 to leave the nucleic acid purification liquid, so that the extraction of nucleic acid is completed.

2. PCR amplification detection module

A) Preparing PCR reaction liquid

After the nucleic acid extraction of the sample is completed, the heating module of an external matched instrument is attached to the outside of the eluent and the detection reagent preservation liquid pool 224, the temperature is raised to 70 ℃ to dissolve paraffin which wraps the PCR detection reagent freeze-dried ball, the dissolved paraffin floats to the uppermost layer, the PCR detection reagent freeze-dried ball is dissolved into the nucleic acid purification liquid, and the PCR freeze-dried ball contains primers for amplifying target fragments, detected probes and other required components, so that the preparation of the PCR reaction liquid is completed.

B) PCR reaction and detection

And setting a corresponding heating program according to the conventional PCR, detecting the PCR reaction liquid through a corresponding detection module of a PCR instrument, and carrying out corresponding fluorescence detection according to the fluorescence of a detection probe of the reaction in the process of the PCR reaction, wherein the process is consistent with the conventional qPCR.

It can be understood that, by combining the nucleic acid purification and detection reaction solution preparation device 100 with a PCR instrument, the utility model actually provides a qPCR detection device for fully-enclosed nucleic acid extraction and amplification, which is equipped with the required nucleic acid extraction and detection reagents, and after the sample is added, fully-enclosed automatic processing is realized, so that the full-automatic detection process of sample input and output is realized, the detection efficiency is high, and the whole process is enclosed, thereby being beneficial to ensuring the accuracy and safety of detection.

The nucleic acid purification and detection reaction solution preparation device 100 of the present utility model is suitable for detection of various clinical samples, such as urine, blood, nasopharyngeal swab, sputum, tissue, feces, etc., and can adjust the reagent formulation and increase or decrease the processing steps according to the specific sample.

The nucleic acid purification and detection reaction solution preparation device 100 realizes the airtight operation of extracting nucleic acid by a conventional magnetic rod method by adopting a silica gel sleeve mode, is a totally-enclosed nucleic acid detection kit, has very good airtight performance, is not communicated with outside air in the use process, can greatly avoid the influence of the external environment on the detection result, and can also protect the health of detection personnel.

The nucleic acid purification and detection reaction solution preparation device 100 can realize the whole-flow operation of sample cracking, nucleic acid adsorption, nucleic acid cleaning, nucleic acid elution and detection reaction solution in a totally enclosed environment through the design of a rotatable main body and the combination of a silica gel sleeve and a magnetic sleeve extraction method, and has the advantages of simple integral structure and operation, and compatibility with various types of samples.

The reagent storage layer 20 of the present utility model includes a plurality of liquid reservoirs 22, which can be configured according to the nucleic acid purification and reaction solution preparation requirements of different samples, and can be compatible with the nucleic acid purification and detection reaction solution preparation requirements of various samples.

The utility model can reduce the arrangement of a flow path switch control valve through the design of the rotary main body, realizes the transfer of magnetic beads in different liquid storage cavities through rotating the structure of the reagent storage main body 21, and provides the nucleic acid purification and detection reaction liquid preparation device 100 for samples, which has simple structure and simple operation.

The nucleic acid purification and detection reaction solution preparation device 100 of the utility model places the eluent and the detection reagent freeze-dried balls in one liquid pool, solves the problem that the nucleic acid eluent needs to be transferred in the preparation process of the detection reaction solution, can omit a liquid path of the device, and avoids the operation of liquid control.

The utility model adopts the freeze-dried ball to store the detection reagent, and wraps a layer of paraffin on the outer surface of the freeze-dried ball of the detection reagent, thereby ensuring that the freeze-dried ball of the detection reagent and the nucleic acid eluent can be placed in a liquid pool, and solving the coexistence problem of the detection reagent and the nucleic acid eluent.

The nucleic acid purification and detection reaction solution preparation device 100 is prepared by adopting an injection molding mode, can realize batch production, and has the advantages of small batch-to-batch difference, high yield and low cost.

The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The foregoing examples only represent preferred embodiments of the present utility model, which are described in more detail and are not to be construed as limiting the scope of the utility model. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the utility model, which are all within the scope of the utility model. Accordingly, the scope of protection of the present utility model is to be determined by the appended claims.

Claims

1. A nucleic acid purification and detection reaction solution preparing apparatus, comprising:

2. The apparatus for preparing a nucleic acid purification and detection reaction solution according to claim 1, wherein the reagent storage layer and the upper cover layer are rotatably connected by a structure of a transmission latch; the range of each rotation angle of the reagent storage layer is 15-90 degrees.

3. The nucleic acid purification and detection reaction solution preparation apparatus according to claim 2, wherein the upper cover further comprises a protrusion having a central groove extending from the upper cover main body, the silica gel cover being fitted in the central groove of the protrusion; the reagent storage main body is provided with a cylindrical structure and a columnar structure extending from the cylindrical structure, the diameter of the columnar structure is smaller than that of the cylindrical structure, and the columnar structure corresponds to the liquid pool one by one.

4. The nucleic acid purification and detection reaction solution preparation apparatus according to claim 3, wherein the plurality of liquid tanks include a magnetic bead storage liquid tank for storing magnetic beads adsorbing nucleic acids, a lysis liquid tank for storing a sample lysis liquid, a washing liquid tank for storing a nucleic acid washing liquid, and an eluent and detection reagent storage liquid tank for storing a nucleic acid eluent and a detection reagent freeze-dried ball wrapped with paraffin; the three cleaning liquid tanks comprise a first cleaning liquid tank, a second cleaning liquid tank and a third cleaning liquid tank which are sequentially arranged.

5. The nucleic acid purification and detection reaction solution preparation apparatus according to claim 4, wherein the magnetic bead storage liquid pool, the lysing liquid pool, the first washing liquid pool, the second washing liquid pool, the third washing liquid pool, and the eluting solution and detection reagent storage liquid pool are arranged at uniform intervals in the circumferential direction of the reagent storage layer in this order.

6. The nucleic acid purification and detection reaction solution preparation device according to any one of claims 1 to 5, wherein a sealing silica gel pad is provided between the upper cover layer and the reagent storage layer.

7. The nucleic acid purification and detection reaction solution preparation apparatus according to any one of claims 1 to 5, wherein the upper lid main body and the reagent storage layer are both prepared by injection molding.

8. The nucleic acid purification and detection reaction solution preparation apparatus according to any one of claims 1 to 5, wherein the nucleic acid purification and detection reaction solution preparation apparatus has an overall length of 35mm, a width of 35mm, and a height of 43mm.

9. The nucleic acid purification and detection reaction solution preparation device according to claim 4 or 5, wherein the sample is sputum, tissue, blood or feces; the detection reagent in the detection reagent freeze-dried ball is PCR, RPA or LAMP reagent.