CN117778181A

CN117778181A - Palm nucleic acid detection device, system and method

Info

Publication number: CN117778181A
Application number: CN202211204476.8A
Authority: CN
Inventors: 沈峰; 张佳杰
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2022-09-29
Filing date: 2022-09-29
Publication date: 2024-03-29

Abstract

The application discloses a palm nucleic acid detection device, which comprises a first module and a second module which can rotate along the same axis, wherein the first module comprises at least one cavity group; the second module comprises a magnetic bead collecting hole for accommodating the magnetic beads and a nucleic acid collecting and detecting cavity; when the two modules rotate around the axle center relatively, the palm nucleic acid detection device at least forms the following steps: a first position, a second position and a third position, wherein the cavity for the cracking reaction, the cavity for the cleaning reaction and the cavity for the elution reaction on the first module respectively form passages with the magnetic bead collecting holes on the second module; and a fourth position, wherein the cavity for the elution reaction forms a channel with the nucleic acid collection detection cavity on the second module. The application also includes a nucleic acid detection system and a method based on the device. The method realizes microminiaturization and integration of the nucleic acid detection equipment and realizes rapid and efficient extraction and enrichment of nucleic acid; has higher sensitivity and accuracy.

Description

Palm nucleic acid detection device, system and method

Technical Field

The present application relates to the field of biochemical reaction analysis, and in particular, to a palm nucleic acid detection device, a palm nucleic acid detection system and a method.

Background

Nucleic acid detection techniques (Nucleic Acid Analysis) can analyze nucleic acids in biological samples. The method has the characteristics of high sensitivity, strong specificity and the like, thereby becoming an important method for biological research and clinical diagnosis. Typical nucleic acid detection techniques include nucleic acid amplification detection techniques, nucleic acid sequencing techniques, nucleic acid hybridization techniques, and the like. Wherein the nucleic acid amplification detection technique in turn comprises a temperature swing nucleic acid amplification technique, such as Polymerase Chain Reaction (PCR); and isothermal nucleic acid amplification techniques, such as loop-mediated isothermal amplification (LAMP), recombinase Polymerase Amplification (RPA), rolling Circle Amplification (RCA), cross Primer Amplification (CPA), strand Displacement Amplification (SDA), helicase-dependent amplification (HDA), and the like. Gene editing technology CRISPR has also begun to be applied to nucleic acid detection.

Nucleic acid detection techniques have important applications in the field of in vitro diagnostics. By detecting the specific gene sequence of the novel coronavirus (SARS-Cov 2), the nucleic acid detection technique can sensitively and accurately diagnose the novel coronavirus (Covid-19). Nucleic acid detection techniques can detect specific gene sequences of pathogens for detection of Sepsis (sepis) and other infectious and infectious diseases. In addition, nucleic acid detection techniques also show great potential for development in the fields of noninvasive prenatal diagnosis (NIPT), tumor-associated diagnosis, tumor early screening, and the like.

The nucleic acid detection technology has important application in the field of detection related to agriculture and animal husbandry. Nucleic acid detection can be used for the relevant detection of infectious diseases of economic animals, such as African swine fever, foot-and-mouth disease, avian influenza and the like. Nucleic acid detection can be used for detection of agricultural crop related diseases. In addition, nucleic acid detection techniques can also be used for detection of pet disease and the environment.

The current nucleic acid detection mode mainly adopts a central laboratory mode. By creating a specialized, compartmentalized laboratory, reagent preparation, sample processing, nucleic acid amplification and product analysis zones are included. Relying on a large number of trained professionals and large machines for operation. The detection flux in the whole process is high, and the requirement of large-scale nucleic acid screening can be met. The construction cost of the central laboratory mode is high, the test period of each batch is longer, the requirements on professional quality of personnel are high, and the construction needs to be carried out in a qualified gene amplification laboratory. These requirements limit the application of the central laboratory model.

In order to meet the demands of non-central laboratory nucleic acid detection, small integrated (Point of Care) nucleic acid detection systems have become an important trend. The system integrates the whole nucleic acid detection process into a small machine, so that the simple and convenient nucleic acid detection can be realized, and no professional staff or a professional laboratory is required. The small integrated nucleic acid detection system reduces the threshold for nucleic acid detection to some extent. At present, a few mature small integrated forms such as foreign Sai pei (Cepheid), film array of biology Mei Liai, alere I of yaban company and the like are put forward in China. For example, the GeneXpert molecular diagnostic system of the Sai-pei company is a fully automatic integrated nucleic acid detection system based on the PCR method, and can specifically detect Mycobacterium tuberculosis and its rifampicin resistance, MRSA (methicillin-resistant staphylococcus aureus), and Clostridium difficile. The system integrates three steps of sample preparation, amplification and detection in a single kit, and automates the same. The person who is trained on the basis can easily complete the whole test.

Existing small integrated nucleic acid detection systems, while meeting to some extent the nucleic acid detection needs of non-centralized laboratories, typically rely on complex and sophisticated instrumentation. Although the price of the device is obviously lower than that of a large-scale instrument in a central laboratory, the price of the device and consumable materials is still higher, and the device and the consumable materials cannot be widely popularized in places such as village and town hospitals, customs ports, disease control sites, side inspection institutions and the like. In addition, such small integrated nucleic acid detection systems cannot well meet emerging application scenarios, such as home nucleic acid detection.

In order to simplify the flow of nucleic acid detection, micro nucleic acid detection systems have been developed. The whole instrument has smaller volume and lower cost, is more convenient for users to operate without professional background, and is beneficial to popularization and use (even household); in combination with the current market demands, a plurality of products for home nucleic acid detection at home and abroad are already introduced or under development. Representative of these are the micro nucleic acid detection systems proposed by Cue Health, lucira Health, detect Inc, etc. The novel coronavirus nucleic acid detection of Cue Health can detect the target N gene and internal standard RNaseP of the novel coronavirus. After the sample enters the detection card, the nucleic acid is released under the ultrasonic action, and the nucleic acid is amplified by adopting the isothermal technology. Wherein, the upstream and downstream primers of the target are respectively marked with biotin and horseradish peroxidase (HRP), and the upstream and downstream primers of the internal standard are respectively marked with digoxin and horseradish peroxidase (HRP), so that the amplified product is provided with a specific mark. The operation process is simple, and after the intelligent mobile phone is connected with the intelligent mobile phone, the whole process can be prompted to operate by the mobile phone App; new generation nucleic acid detection technology (New ERA) which is developed independently by Souzhou first reach Gene technology Co., ltd, and matched with micro nucleic acid fluorescence detector (Gene mirror) ) An integrated solution for detecting the nucleic acid of the New ERA of the first gene is established. The current micro nucleic acid detection system is simple, but the detection performance is still to be improved. Many products are only thermally cracked in situ, simply without the process of nucleic acid extraction purification enrichment. This results in insufficient sensitivity, unstable experimental results, and poor repeatability of the system detection.

In summary, the main disadvantages of the existing micro nucleic acid detection products are:

1. insufficient detection performance

Insufficient detection performance is the biggest problem of current miniature nucleic acid detection systems. In order to simplify the whole nucleic acid detection step and reduce the volume of the instrument, many products are simply subjected to in-situ thermal cracking, and no nucleic acid extraction, purification and enrichment process is carried out, so that the detection sensitivity is insufficient. In addition, the results of the experiment are not stable enough and the repeatability is poor. The detection of the occurrence of "false positives", "false negatives" does not result in a confidence level.

2. High instrument cost

The cost of the instrument becomes a very important contributor to the micro nucleic acid detection system. The excessive price makes the micro nucleic acid detection system convenient, but has no strong attractive and popularization prospect. The current foreign micro nucleic acid detection system has higher cost.

3. Time of detection

The detection time is a very important consideration for a micro nucleic acid detection system. Some detection systems detect detection times in excess of 60 minutes. This makes it difficult for the micro nucleic acid detection system to present the results to the user.

Therefore, there is a need to develop a micro nucleic acid detecting device that can achieve the advantages of system size, detection cost, convenience in operation, and nucleic acid detecting performance, and a system and a method for using the same.

Disclosure of Invention

The purpose of the application is to provide a miniaturized and highly integrated palm nucleic acid detection device, a system and a method thereof, which shorten the detection reaction time on the basis of realizing the miniaturization of nucleic acid detection equipment, rapidly present the detection result, and simultaneously, the miniaturized system matched with the device can be produced at low cost.

In one aspect of the application, a palm nucleic acid detection device is disclosed, comprising a first module and a second module which can rotate along the same axis, wherein the first module comprises at least one cavity group, and the cavity group at least comprises a cavity for a cracking reaction, a cavity for a cleaning reaction and a cavity for an eluting reaction; the second module comprises a magnetic bead collecting hole for accommodating the magnetic beads and a nucleic acid collecting and detecting cavity; when the two modules rotate around the axle center relatively, the palm nucleic acid detection device at least forms the following steps:

A first position, wherein a cavity used for a cracking reaction on the first module and a magnetic bead collecting hole on the second module form a passage;

a second position, wherein a cavity used for cleaning reaction on the first module and a magnetic bead collecting hole on the second module form a passage;

a third position, wherein a cavity used for eluting reaction on the first module and a magnetic bead collecting hole on the second module form a passage;

in the fourth position, the cavity for the elution reaction on the first module forms a passageway with the nucleic acid collection detection cavity on the second module, at which time the eluent containing purified nucleic acid flows into the nucleic acid collection detection cavity of the second module.

In a preferred embodiment, in the first position and/or the second position and/or the third position, the magnetic beads are dispersed into the cavity containing the solution of the first module under the action of an external force, and then fall into the magnetic bead collecting hole of the second module again.

In a preferred embodiment, the cavity group of the first module further comprises a cavity for receiving a control reagent.

In a preferred embodiment, the volume of the magnetic bead collection well is 10% or less of the volume of the cavity used for the elution reaction.

In a preferred embodiment, the external force that causes the magnetic beads to move up and down between the first module and the second module is selected from any one or a combination of magnetic force and gravity.

In a preferred embodiment, the nucleic acid collection detection cavity of the second module is an amplification tube.

In a preferred embodiment, the nucleic acid collection detection chamber comprises a nucleic acid detection reagent selected from any one of the following: variable temperature amplification reagents, isothermal amplification reagents, hybridization-based nucleic acid detection reagents.

In a preferred embodiment, the palm nucleic acid detecting device comprises a plurality of cavity groups, and the second module comprises a plurality of magnetic bead collecting holes and a plurality of nucleic acid collecting detecting cavities corresponding to the first module.

In another aspect of the present application, a palm nucleic acid detecting device system based on any one of the above palm nucleic acid detecting devices is disclosed, further comprising: the base, the base is a sealed housing of uncapping type, and inside cavity is provided with and is equipped with the buckle with palm nucleic acid detecting device adaptation in order to fix it, the base includes: control module, heating module, magnetic force module, wherein

The magnetic force module is configured to be turned on or off based on the demand of magnetic bead enrichment or oscillation;

the heating module comprises a piezoelectric ceramic piece and is configured to heat, cool or preserve heat the nucleic acid collection and detection cavity; the temperature sensor is used for detecting the temperature of the piezoelectric ceramic piece and transmitting signals to the control module;

The control module is configured to receive signals from the heating module and/or the magnetic module and further control the operating state of the module based on the control signals; and performing data processing on the signals in the acquired palm nucleic acid detection device, thereby obtaining an analysis result.

In a preferred embodiment, the device further comprises an optical detection module configured to illuminate with excitation light to cause fluorescence of the substance within the nucleic acid collection detection chamber and to record the resulting fluorescence signal.

In a preferred embodiment, a data transmission module is also included, the data transmission module including at least the capability to communicate wirelessly over short distances with consumer electronic devices including any one or more of smartphones, personal computers, smartwatches, and tablet computers.

In yet another aspect of the present application, a method for detecting palm nucleic acid is also disclosed, comprising the steps of:

(S1) taking the palm nucleic acid detection system, wherein a lysis solution, a cleaning solution and an eluent are respectively preset in a cavity for a lysis reaction, a cavity for a cleaning reaction and a cavity for an elution reaction of the first module; a nucleic acid detection reagent is preset in the nucleic acid collection detection cavity of the second module; after tearing the membrane, adding a sample into the cavity for the cleavage reaction;

(S2) enabling the palm nucleic acid detection device to be located at a first position, and vibrating the palm nucleic acid detection device; after standing for a moment, placing the palm nucleic acid detection device on a base, and applying magnetic force to the magnetic bead collection holes;

(S3) twisting the palm nucleic acid detection device to a second position, and vibrating the palm nucleic acid detection device; after standing for a moment, placing the palm nucleic acid detection device on the base, and applying magnetic force to the magnetic bead collection hole of the second module;

(S4) twisting the palm nucleic acid detecting device to a third position, vibrating the palm nucleic acid detecting device; after standing for a moment, placing the palm nucleic acid detection device on the base, and applying magnetic force to the magnetic bead collection hole of the second module;

(S5) twisting the palm nucleic acid detecting device to a fourth position; vibrating the palm nucleic acid detection device;

(S6) using a heating module of the base, performing nucleic acid amplification on the nucleic acid collection detection cavity for 30 minutes;

and (S7) carrying out data processing on the experimental result to obtain a qualitative or quantitative detection result.

The application has the following technical effects:

1. the nucleic acid extraction method of the micro-fluidic chip and the magnetic beads are combined to realize the rapid and efficient extraction and enrichment of nucleic acid; compared with other methods such as thermal cracking, the method for extracting nucleic acid by using the magnetic beads has higher sensitivity and accuracy;

2. The flow path is switched for multiple times through the change of the relative positions of the upper chip and the lower chip, and finally the whole flow of nucleic acid detection is finished through simple operation, so that the operation process is simple, quick and convenient, and the non-professional personnel can finish the detection;

3. a variety of detection schemes are provided. The reaction results can be directly observed by naked eyes through the pH sensitive reagent. And can be detected by the optical module. The reagent can be stored for a long time through structural design, so that the problems of storage and transportation of some biological reagents are solved;

4. the integrated nucleic acid detection system with matched miniaturization comprises a nucleic acid amplification function, a stable heating module and an optical detection module, can realize the amplification and signal reading of nucleic acid, and finally presents the detection result to a user;

5. the whole product can be processed by injection molding, so that a large amount of cost is saved, the volume of the detection consumable and the detection instrument is small, the price is low, the operation is convenient, and the popularization of the nucleic acid detection are facilitated.

In the present application, a number of technical features are described in the specification, and are distributed in each technical solution, which makes the specification too lengthy if all possible combinations of technical features (i.e. technical solutions) of the present application are to be listed. In order to avoid this problem, the technical features disclosed in the above summary of the present application, the technical features disclosed in the following embodiments and examples, and the technical features disclosed in the drawings may be freely combined with each other to constitute various new technical solutions (which should be regarded as having been described in the present specification) unless such a combination of technical features is technically impossible. For example, in one example, feature a+b+c is disclosed, in another example, feature a+b+d+e is disclosed, and features C and D are equivalent technical means that perform the same function, technically only by alternative use, and may not be adopted simultaneously, feature E may be technically combined with feature C, and then the solution of a+b+c+d should not be considered as already described because of technical impossibility, and the solution of a+b+c+e should be considered as already described.

Drawings

FIGS. 1 to 3 are perspective, bottom, and side views of a palm nucleic acid detecting apparatus according to one embodiment;

FIGS. 4 to 6 are perspective, bottom, and top views of an upper chip of the palm nucleic acid detecting apparatus according to one embodiment;

FIGS. 7 to 9 are perspective, bottom, and top views of a lower chip of the palm nucleic acid detecting apparatus according to one embodiment;

FIG. 10 is a deformable material of a palm nucleic acid detection device according to one embodiment;

FIG. 11 shows a schematic diagram of the apparatus after the first and second modules are combined;

FIGS. 12A, 12B show exploded views of the device;

FIGS. 12C, 12D show schematic views of an apparatus according to another embodiment;

FIG. 13 is a schematic diagram of the principle of operation of the palm nucleic acid detecting apparatus in a first position according to one embodiment;

FIG. 14 is a schematic diagram illustrating the principle of operation of the palm nucleic acid detecting apparatus in a second position according to one embodiment;

FIG. 15 is a schematic diagram illustrating the principle of operation of the palm nucleic acid detecting apparatus in a third position according to one embodiment;

FIG. 16 is a schematic diagram showing the operation of the palm nucleic acid detecting apparatus according to one embodiment in a fourth position;

FIG. 17 is a unitary structure of a base according to one embodiment;

fig. 18 is a schematic diagram of a submount placed in a microfluidic chip according to an embodiment;

FIG. 19 is a schematic view of the internal structure of a base according to one embodiment;

FIG. 20 is a schematic view of a heating module in a base according to one embodiment;

FIG. 21 is an overall block diagram in a base according to another embodiment;

FIG. 22 is an internal schematic view in a base according to another embodiment;

FIGS. 23, 24 are top, plan views of a heating module in a base according to another embodiment;

FIG. 25 is an exploded view of the upper and lower chips according to example 1;

fig. 26 is a top-bottom chip assembly diagram according to embodiment 1;

FIGS. 27A-27D are top, bottom, perspective, and side views of an upper chip according to embodiment 1;

fig. 28A-28D are perspective, side, bottom, top views of a lower chip according to embodiment 1;

FIG. 29 is a graph comparing CT values for multiple new coronavirus tests using the apparatus and system of example 1;

FIG. 30 is a chip on device according to use example 2;

fig. 31 is a schematic diagram of the upper and lower chips of the apparatus according to use example 2.

FIG. 32 is a schematic diagram showing the operation principle of the palm nucleic acid detecting apparatus according to example 2 in the first position;

FIG. 33 is a schematic view showing the operation principle of the palm nucleic acid detecting apparatus according to example 2 in the second position;

FIG. 34 is a schematic view showing the operation principle of the palm nucleic acid detecting apparatus according to example 2 in the third position;

FIG. 35 is a schematic view showing the operation principle of the palm nucleic acid detecting apparatus according to example 2 in the fourth position;

FIG. 36 is a schematic view showing the operation principle of the palm nucleic acid detecting apparatus according to embodiment 2 in the fifth position;

reference numerals illustrate:

1-sticking a film to a sample hole; 2-upper surface sealing film;

3-chip loading; 31-lysate wells; 32-cleaning liquid holes; 33-eluent wells; 34-control reagent wells;

4-lower chip; 41-magnetic bead collection well; 42-purifying nucleic acid passing holes; 43-contrast agent passage hole; 44-spacing structural locating areas; 45-limiting the rotation angle of the upper chip; 46-fixing the clamping position of the lower chip; 47-chip mounting clip;

5-amplification tube; 501-nucleic acid collection detection amplification tube; 502-control reagent amplification tube;

6, a base; 601-upper cover buckle; 602-a chip fixing region; 603-a switch; 604-power supply; 605-a display; 61-a magnetic force module; 62-a heating module; 63-an optical detection module; 64-a control module; 65-a fluorescence detection module;

631-LED lamp beads; 632-an excitation light filter; 633-lens group; 634-a detection filter; 635-ceramic heater plate; 636-a detection filter;

7-deformable material.

Detailed Description

The inventor of the application has conducted extensive and intensive studies and has proposed a palm nucleic acid detection system and a method of using the same, comprising a microfluidic chip-based and palm nucleic acid detection device which can complete the whole flow of nucleic acid extraction and nucleic acid amplification and detection of "sample in-out", without opening the cover in the middle ".

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be understood by those skilled in the art that the claimed invention may be practiced without these specific details and with various changes and modifications from the embodiments that follow.

The following outline describes some of the innovative points of the embodiments of the present application:

the palm nucleic acid detection device provided by the invention is based on a microfluidic chip and comprises two modules (shown in figures 1-3) which can relatively rotate along the same axis. The two modules may be designed as cylinders, squares or any other shape. The two modules are respectively designed with a preset and liquid-storing cavity and/or micro-pits and/or micro-holes, and are also provided with functional cavities such as amplification tubes. These two modules may be manufactured by a variety of means including, but not limited to, 3D printing, computer Numerical Control (CNC) machining, injection molding machining, and the like. The two modules may be made of the same or different materials, and the materials may be selected from any one or several of the following: plastic, rubber, wood materials, metal, glass, quartz, silicon-based materials, ceramics, and the like. The surfaces of the two modules may be subjected to hydrophilic or hydrophobic surface modification treatments.

The upper module of the present invention is a first module, hereinafter referred to as "upper chip", and includes a series of cavity groups for placing reagents. A typical set of cavities includes a lysate chamber 31, a wash liquid 32 chamber, and an eluent chamber 33. In another alternative embodiment, the cavity set further includes a cavity for receiving a control reagent, i.e., a control reagent well 34 (shown in FIG. 6). The cavities may be arranged in a track with the center of the circle on the same circle or in a track with a non-circle. The control reagent wells may also be located on a circumferential trajectory at unequal distances from the center from the eluent reagent wells, as shown in FIG. 12C. These cavities may be of the same or different volumes. Typically, the largest volume cavity is used for the combination in which the lysate reaction occurs. The volume of the chamber may be 1 microliter to 100 milliliters. In yet another embodiment, multiple samples can be processed simultaneously on the same chip, as shown in FIG. 12D, the chip contains multiple sets of cavities (A1, B1, C1, D1), (A2, B2, C2, D2) including lysate wells, wash wells, eluent wells, as shown in FIG. 12D. Such an upper chip comprising multiple sets of cavities allows 2 and more samples to be processed simultaneously on the same chip. Fig. 4-6 show perspective, bottom, and top views, respectively, of the upper die of a preferred embodiment.

The lower module of the present invention is a second module, hereinafter referred to as "lower chip", comprising one or more micro-chambers, the kinds of which include at least a magnetic bead collection well 41, and may further include a purified nucleic acid passing well 42, a control reagent passing well 43. The lower parts of the pure nucleic acid passing hole and the control reagent passing hole are respectively connected with an amplifying tube 5 for collecting and detecting nucleic acid. The microcavities of the lower chip may be of different shapes. The volume of the microcavity may range from 1 picoliter to 10 milliliters. The arrangement of these microcavities should satisfy: when the upper and lower chips are combined and rotated with each other about the same axis, the respective cavities of the upper chip must be vertically aligned with the magnetic bead collection holes, i.e., the magnetic bead collection holes should be arranged on the "track" through which the respective cavities of the upper chip pass. Meanwhile, the purified nucleic acid passing hole, the control reagent passing hole must be arranged in the "track" passed by the eluent hole of the upper chip. Under the above premise, the micro-chambers of the lower chip 5 may be arranged on a circular track or a plurality of circular tracks or non-circular tracks. Fig. 7-9 show perspective, bottom, and top views, respectively, of a lower chip of a preferred embodiment.

The contact surfaces of the two modules of the device can be tightly attached with the upper chip 3 and the lower chip 4 by adding deformable materials. Wherein the soft material may be, but is not limited to, plastic, rubber, TPU, TPE, liquid silicone, etc. The surface of the deformable material may be hydrophilized or hydrophobically modified. The surface of which may also be added with a lubricating phase for lubrication. The surface of which may also be added with a sealing phase for improving sealability, such as a sealing silicone grease. The deformable material may be added to the upper chip or to the lower chip. The deformable material can also be designed into a sheet shape, and is provided with through holes or cavity structures matched with the hole sites of the upper chip and the lower chip. This sheet-like deformable material may be secured to the upper (and) or lower (and) chips. Fig. 10 is an embodiment of a sheet of deformable material.

The device is assembled from an upper chip 3 and a lower chip 4. The upper and lower chips are fixed by means of mutually engaged structures, such as screws or snaps. The upper chip and the lower chip have one surface which can be in relatively close contact. The deformable material described above may be placed intermediate the upper and lower chips to help provide intimate contact. Fig. 11 shows a preferred embodiment of the assembled device. Fig. 12A and 12B show an exploded view of the device and the alignment of its hole sites, with the upper chip, deformable material, and lower chip in that order from top to bottom.

For the purpose of making the objects, technical solutions and advantages of the present application more apparent, embodiments of the present application will be described in further detail below with reference to the accompanying drawings.

The palm nucleic acid extraction device according to the present invention employs a nucleic acid extraction method based on magnetic beads. The working principle of the nucleic acid extraction method based on the magnetic beads is as follows:

(1) The sample is added to the pre-set reagent device, the sample is lysed and the nucleic acids are released into the solution. The means of pyrolysis include, but are not limited to, thermal pyrolysis, pyrolysis liquid pyrolysis, ultrasonic pyrolysis, physical pyrolysis, chemical pyrolysis, biological pyrolysis, and the like, or combinations of these means.

(2) The magnetic beads capture nucleic acids in solution.

(3) And cleaning the magnetic beads by using a cleaning solution.

(4) The eluting solution elutes the nucleic acid from the magnetic beads into the aqueous solution.

(5) The aqueous solution containing the extracted nucleic acid is mixed with a nucleic acid detection reagent to detect the target nucleic acid sequence.

The washing step of (3) above may be omitted in the actual detection flow. One or more of the lysate, the cleaning solution, the eluent and the nucleic acid detection reagent are pre-buried in the corresponding chamber of the microfluidic chip. Alternatively, the known nucleic acid sequence as a reference may be placed as a control reagent in another chamber beside the sample and then separately mixed with the nucleic acid detection reagent of (5).

The nucleic acid detection reagent can be temperature-variable nucleic acid amplification, isothermal nucleic acid amplification, nucleic acid hybridization, direct detection of nucleic acid, physical change of a nucleic acid initiation system, CRISPR and the like. The nucleic acid detection may be real-time detection or end-point detection. The detection method can be naked eye detection or can be assisted by an auxiliary detection instrument.

The following describes the working principle of the palm nucleic acid detecting device of the present invention:

(2) The device is placed at a first position (namely a cracking position), and a passage is formed between a cracking liquid hole of the upper chip and a magnetic bead collecting hole of the lower chip. In the first position, the magnetic beads are dispersed into the solution of the lysis solution hole of the upper chip by external force, and the nucleic acid in the solution is captured.

After a while, in the first position (cleavage position), the beads re-enter the bead collection well of the lower chip under the influence of additional droplet external forces (e.g., magnetic force or gravity).

(3) Under the action of external force (such as manual torque force), the upper chip is moved to a second position (namely a cleaning position) relative to the lower chip, and a passage is formed between the cleaning liquid hole of the upper chip and the magnetic bead collecting hole of the lower chip. In the second position (i.e., the washing position), the magnetic beads in the magnetic bead collection hole of the lower chip are dispersed into the solution in the washing liquid hole of the upper chip by an external force, and the nucleic acid in the solution is washed.

After a while, in the second position (washing position), the beads are again introduced into the bead collection well of the lower chip by additional droplet external force (e.g., magnetic force or gravity).

(4) Under the action of external force (such as manual torque force), the upper chip is moved to a third position (i.e. elution position) relative to the lower chip, and the eluent hole of the upper chip and the magnetic bead collecting hole of the lower chip form a passage.

In the third position (i.e., elution position), the magnetic beads in the magnetic bead collection well of the lower chip are dispersed by external force into the solution in the eluent well of the upper chip, and the nucleic acid is eluted into the solution. In the third position (elution position), the beads enter the bead collection well of the lower chip under the influence of additional droplet external forces (e.g., magnetic or gravitational forces).

(5) Under the action of external force (such as manual torque force), the upper chip is moved to a fourth position (collecting-detecting position) relative to the lower chip, and the eluent hole of the upper chip and the nucleic acid detecting hole of the lower chip form a passage.

In the fourth position (i.e., the collection-detection position), the solution in the eluent hole of the upper chip flows into the amplification tube through the purified nucleic acid passing hole of the lower chip under the action of external force, and is mixed with the nucleic acid detection reagent.

In the fourth position (collection-detection position), the detection reaction is performed in the purified nucleic acid passing well (or amplification tube) of the lower chip.

In a preferred embodiment, the relative positional movement of the upper and lower chips mentioned in (2), (3), (4), (5) of the above principle of the present invention may be accomplished manually or by auxiliary instrument means.

In a preferred embodiment, the external force for dispersing the magnetic beads mentioned in the above principles (2), (3) and (4) of the present invention may be applied manually or may be accomplished by an auxiliary instrument.

In a preferred embodiment, the magnetic force for collecting magnetic beads mentioned in the above-mentioned principles (2), (3) and (4) of the present invention may be a permanent magnet or an electromagnet.

In a preferred embodiment, the sample lysis of (1) in the above principle of the invention may be done on-chip or in a container outside the chip.

In a preferred embodiment, the nucleic acid detecting reagent according to the above principle (5) of the present invention may be a liquid or a dried or lyophilized reagent.

In a preferred embodiment, the nucleic acid detection reagent of (5) in the above principle of the present invention may be a temperature-variable amplification reagent such as PCR; isothermal amplification reagents, such as LAMP, RPA, RCA, HDA, NASBA, TMA, etc., are also possible; CRISPR is also possible; hybridization-based nucleic acid detection reagents are also possible.

In a preferred embodiment, the nucleic acid detecting reaction of (5) in the above-described principle of the present invention may be a color change in the visible wavelength range by the reaction process or the reaction end point; but also signal variations of non-visible wavelengths such as fluorescence or raman etc.

In a preferred embodiment, the nucleic acid detection reaction of (5) of the present invention uses a hot melt wax for sealing treatment, which solves the problem of contamination of the micro nucleic acid detection system due to the absence of a hot cap.

After the chip operation is completed, the device of the invention can be placed in a device capable of controlling temperature to perform isothermal amplification. Such devices include, but are not limited to, small temperature control devices, ovens, water baths, metal baths, and the like.

The method has a certain volume of liquid residue. This volume is approximately equal to the volume of the magnetic bead collection well of the lower chip minus the volume of the magnetic beads. This residual liquid can be carried from upstream to downstream in the process flow. For example, a small amount of residual liquid of the lysate may be carried into the cleaning liquid; for another example, a small amount of liquid may be carried from the cleaning liquid location to the elution location, thereby entering the eluent. Liquid residues in the eluate (lysate and/or wash solution) may negatively affect the amplification detection of the nucleic acid (e.g., affect the sensitivity and/or specificity of the detection). Therefore, it is necessary to control the volume of the magnetic bead collection well of the lower chip so that it does not exceed a certain proportion of the liquid reagent in the reagent well of the upper chip. The volume of the magnetic bead collection well of the lower chip for magnetic bead collection needs to be adjusted according to different nucleic acid extraction reagents and different nucleic acid amplification detection reagents. The volume of the magnetic bead collection well of the lower chip is preferably less than 10% of the eluent well volume.

Nucleic acid extraction reagents (lysate, wash, eluate) may be directed against DNA or RNA; the nucleic acid extraction reagent may be directed to different samples, including but not limited to blood, urine, saliva, swabs, and the like.

On the other hand, the invention also provides a base which can be matched with the palm nucleic acid detection device for use. The base is provided with a heating module, a control module, a magnetic force module and a battery or a power supply.

One basic embodiment of the base is shown in fig. 19, where the base 6 enclosure is of flip-top design, where the upper and lower shells are secured by a key top snap 601. The base cavity includes a chip holding area 602 with a permanent magnet disposed at the bottom for magnetically coupling to the magnetic beads inside the chip. In an alternative, a magnet is provided at the bottom of the region.

The inner wall is provided with a groove for limiting the nucleic acid extraction device. The amplification tubes of the chip, control reagent amplification tubes (e.g., positive quality control amplification tubes), are contacted with a heating module. The heating module in the base is shown in fig. 20. When the chip is used for extracting nucleic acid, the upper cover and the lower cover are opened; when the extraction is completed and the subsequent heating amplification reaction is carried out, the upper cover and the lower cover are closed, so that the relative sealing of the internal environment is ensured, and the stability of the heating amplification reaction is ensured. An image display screen in the form of a user interface template is arranged below the outer wall of the base and is provided with one or more data input fields for user input (such as current temperature, heating time) including temperature, heating time and display of electric quantity. When the detection result is checked, the cover can be opened again to take out the chip, and the result can be judged by visually checking the color change of the amplification tube.

The heating module of the base of the invention adopts a piezoelectric ceramic heating plate for heating. In other alternative embodiments, the heating element may also be a semiconductor device that implements temperature control based on the Peltier effect. Temperature monitoring is performed by the temperature sensor pt1000, and temperature control is performed in a feedback mode. The temperature can be basically maintained at a relatively stable value through circuit design, and the temperature requirement of a reaction system such as LAMP, RPA, RAA for isothermal nucleic acid amplification is met. The heating module may also be designed for temperature-variable amplification of PCR.

The control module of the present invention is responsible for controlling the heating module 62 while displaying the amount of electricity and timing the reaction time. The control module is configured to receive signals from the heating module and/or the magnetic module and further control the operating state of the module based on the control signals; and performing data processing on the signals in the acquired palm nucleic acid detection device, thereby obtaining an analysis result.

In a preferred embodiment, the base further comprises an optical detection module 63, which may be integrated with the heating module. As shown in fig. 21 to 24, the optical detection module includes an LED lamp 631, an excitation light filter 632, a lens group 633, a heating module 62, a detection filter 634, and a fluorescence detection module 65 including an imaging device. In order to avoid the interference of reflected light to the imaging device, the LED lamp beads are not perpendicular to the plane where the detected microfluidic chip is located, but are at a certain angle. The excitation light filter is arranged on the light path of the LED lamp beads and is used for irradiating the amplification tube with a specific wavelength (for example, 480 nm). The amplified liquid emits fluorescence after being irradiated by excitation light. The detection filter is arranged in front of the fluorescence detection module. The fluorescence is imaged by the imaging device through a detection filter, for example, at a center wavelength of 520 nm. The imaging device can be mobile photographing devices such as a mobile phone, a cloud camera or a digital camera, and can also be a photoelectric chip with an imaging function, and the imaging device can be used for photographing the microfluidic chip at intervals for real-time fluorescence detection. The imaging device may also contain manual or automatic focusing means. The detector for fluorescence collection is vertically arranged with the diode of the light source, so that the interference of the two light rays is avoided. By collecting the fluorescent signal, whether the virus exists can be intuitively judged. And through the real-time collection of signals, the amplification curve of the whole nucleic acid can be drawn according to the signals, so that the viral load is roughly judged, and further semi-quantitative detection is realized. Wherein the positive result has high fluorescence intensity and the negative result has weak fluorescence intensity. The fluorescence channel detected by the fluorescence detection module can be FAM, cy5, HEX, ROX, JOE, or the like. Depending on the design of the primers and probes, single channel detection, or multi-channel detection may be used.

The control module can judge the overall result of the reaction according to the result of the fluorescence detection module, and finally the result is displayed to the detection personnel through digital display.

In a preferred embodiment, the base may also include a mechanical control module that integrates an automated protocol for automated nucleic acid extraction. The mechanical control module comprises a multipurpose motor which is used for providing power for rotating the upper chip and the lower chip, controlling the magnetic force module and oscillating.

The inner cavity of the base is provided with a part of mechanical device, so that the upper chip and the lower chip can rotate through a motor driving rotating shaft, the chips can rotate by a fixed angle, and the alignment of specific areas of the upper chip and the lower chip is realized.

The lower part of the second module is attached to the base, and the magnetic force module controls current to pass through programming, so that the control on the magnetism of the magnet is realized, and the magnet can be magnetized when the magnet is required to be enriched, and is not magnetized when the magnet is oscillated and resuspended.

The step of vibration and uniform mixing adopts motor vibration. The magnetic beads can be rapidly resuspended and dispersed in a liquid system, so that the extraction speed is increased.

The mechanical control module allows the system to be automated, thereby optimizing ease of use. When in use, a user only needs to put the sampled sample into the sample splitting area, and then the sealing film is attached. The machine then starts automatic nucleic acid extraction. The automated system workflow is as follows: firstly, the electromagnet is not electrified, and the motor firstly vibrates, so that on one hand, the lysate fully lyses viruses, and internal nucleic acid is released. The other side is oscillated to disperse the magnetic beads and combine with the nucleic acid in the sample. The shaking lasts about 20 seconds. Then the electromagnet is electrified to generate magnetism so that the magnetic beads are enriched and attracted to a magnetic bead collecting area below the sample cracking area.

Followed by a cleaning step. The electric rotating shaft rotates the upper chip clockwise on the base, so that the cleaning liquid holes of the upper chip are aligned with the magnetic bead collecting area. And the electromagnet is turned off. Starting vibration to vibrate, and vibrating to enable the magnetic beads to be fully dispersed, so that the cleaning liquid is fully mixed and contacted with the magnetic beads, and the specificity of extraction is guaranteed. Shaking for about 10 seconds. And then the electromagnet is started to enable the magnetic beads to be enriched and absorbed to fall back to the magnetic bead collecting holes below the cleaning liquid holes.

Followed by a sample elution step. The motor outputs torque to cause the mechanism to rotate the upper chip clockwise on the base such that the eluent aperture of the upper chip is aligned with the magnetic bead collection aperture. And the electromagnet is turned off. Starting vibration to vibrate, and enabling the magnetic beads to be fully dispersed into the eluent, so that nucleic acid in the magnetic beads is fully eluted, and meanwhile, positive control reagents fall into a control reagent amplification tube below through liquid in the hole. The shaking lasts about 20 seconds. And then the electromagnet is started to enrich the magnetic beads and suck the magnetic beads into a magnetic bead collecting hole below the nucleic acid eluting hole.

Finally, collecting the sample. The motorized spindle rotates the upper chip counter-clockwise on the base such that the upper chip nucleic acid eluting region is aligned with the sample collection region. And then the vibration module vibrates, and the liquid in the vibration nucleic acid eluting area fully falls into the test tube of the sample collecting area below.

Thus, the whole process takes no more than 90 seconds after the nucleic acid is extracted by the magnetic bead method based on the system of the invention.

The device is placed on a base to be heated. Opening a ROX channel for fluorescence detection at 65 ℃ for 25 seconds, and exposing for 1 second; the FAM channel was then opened for fluorescence detection with an exposure time of 0.5 seconds. The collected fluorescence signal intensities of ROX and EvaGreen (FAM channel) can be subjected to subsequent analysis to obtain corresponding quantitative results.

The average intensity of fluorescence signals of each microwell in each cycle can be automatically tracked and analyzed by a program, and a real-time fluorescence curve of each microwell is drawn. The quantitative results have good consistency under the sample condition of the same concentration, fluorescence of the micropores is monitored in real time, false positive micropores can be effectively removed according to an amplification curve, and the high-sensitivity and high-specificity quantitative monitoring is realized by combining the digital results of the microfluidic chip.

In a preferred embodiment, the base further comprises a data transmission module. The data transmission module transmits the data acquired by the chip to terminals not limited to mobile phones, tablet computers and notebook computers. The transmitted data are not limited to the sample amplification curve, amplification time, etc. collected by the chip. Meanwhile, the amplified data can also be connected with a nucleic acid result query platform such as 'random code', 'health care', and the like, so that a user can do nucleic acid detection at home without going out. The transmission mode may be bluetooth or wireless.

In order to better understand the technical solutions of the present application, the following description is given with reference to a specific example, in which details are listed mainly for the sake of understanding, and are not meant to limit the scope of protection of the present application.

Example 1 detection of New coronavirus by Manual operation Using PH color change

The first embodiment of the present application relates to a palm nucleic acid detecting apparatus, which realizes extraction of nucleic acid by a magnetic bead method by simple hand-held operation. By means of isothermal amplification, a matched nucleic acid amplification and detection machine reads results, and real POCT instant detection is achieved.

As shown in fig. 25, the palm nucleic acid detecting device comprises seven parts, namely a sample hole adhesive film 1, an upper surface sealing film 2 (aluminum film), an upper chip 3, a deformable material 7 (silica gel gasket), a lower chip 4, a nucleic acid collecting detection amplification tube 501 and a control sample amplification tube 502. The aluminum film is attached to the upper chip before use, the silica gel gasket is attached to the upper side of the lower chip, and the nucleic acid collecting and detecting amplification tube and the control sample amplification tube are tightly connected with the lower chip. The upper core chip is fixed together through a buckle structure, and the proper distance between the upper core chip and the lower core chip is kept.

The upper chip is mainly responsible for storing different reagents, the lower chip is responsible for bearing magnetic beads, and the function of nucleic acid extraction is realized through rotation. The upper chip mainly comprises four areas, namely a sample lysate hole, a cleaning night hole, an eluent hole and a positive quality control hole (namely a control reagent hole). Each area of the upper chip adopts a closing-in shape design, so that a magnetic bead sample can fall into a magnetic bead collecting area below better. Top, side, top, and isometric views of the upper die are shown in fig. 27A-27D, respectively.

An isometric view, side view, bottom view, top view of the lower chip is shown in fig. 28A-28D. The lower chip is attached to the silica gel pad, and the whole lower chip consists of 7 parts, namely a magnetic bead collecting hole 41, a purified nucleic acid passing hole 42 (corresponding to a nucleic acid collecting and detecting amplification tube hole), a contrast reagent passing hole 43 (corresponding to a contrast reagent amplification tube hole), a spacing structure positioning area 44, an upper chip rotating angle limit 45, a lower chip fixing clamp 46 and a chip mounting clamp 47. Wherein the purified nucleic acid passes through the hole and the contrast agent passes through the hole and is connected with the amplification tube below the hole, thereby facilitating the collection of liquid. The space structure positioning areas are four in number and distributed around the lower chip. The top, side, top, and isometric views of the lower core plate are shown below.

The N gene of the corresponding novel coronavirus is detected by the nucleic acid detection device, and the sequence is as follows:

the composition of the lysate in the lysate well is: 30g guanidine isosulfate was dissolved in 40mL of 0.1M Tris buffer, followed by 8.8mL of 0.2M EDTA (pH=8.0) and 0.65mL of Triton X-100 (pH=6.4).

The composition of the cleaning solution in the cleaning solution hole is 70% ethanol aqueous solution.

The composition of the eluent in the eluent hole is non-nucleic acid water.

The magnetic Beads in the magnetic bead collection well were Magbinding Beads (Zymo Research).

The chip on the device is formed by injection molding, the material of the chip is PP, the pore volume of the lysate is 2.5mL, the volume of the cleaning hole is 1mL, and the volume of the eluting hole is 0.2mL. The lower chip is made of black PC material and is also processed by injection molding. The volume of the magnetic bead collection well on the lower chip was 1. Mu.l.

1000 microliters of lysate was pre-placed in the lysate wells, 500 microliters of wash solution was pre-placed in the wash solution wells, 100 microliters of water was pre-placed in the eluent wells, and 10 microliters of magnetic beads were pre-placed in the lysate wells and pre-mixed with the lysate.

The nucleic acid collecting and detecting amplification tube below the lower chip is pre-set with freeze-dried amplification reagent and low melting point paraffin. The freeze-dried amplification reagent comprises an isothermal amplification PH sensitive reagent system and 6 LAMP primers. The lower amplification reagent system was first calculated from liquid and contained 7. Mu.L of dNTPs at a concentration of 10mM, 0.8. Mu.L of 50. Mu.M (LF), 0.8. Mu.L of 50. Mu.M (LB), 0.2 of 50. Mu.M (F3), 0.2. Mu.L of 50. Mu.M (B3), 3.6. Mu.L of 10 Xisothermal amplification reagent, 3. Mu.L of 100mM magnesium sulfate, 2.5. Mu.L of 20mg mL-1BSA solution, 1mg of 501mg mL chromogenic reagent, 3mL of Bst 2.0WarmStartL, and containing polymerase 2 2. Betaine solution at a concentration of 6 mM.

The LAMP amplification primer sequences are:

TABLE 1 LAMP amplification primer sequences

Paraffin wax (Sigma-Aldrich) has a melting point of 53-58 degrees, about 2g. Together with the lyophilized reagent, is added to the nucleic acid collection detection amplification tube.

The reference gene P72 is in the amplification tube of the control reagent (namely the positive amplification tube), the amplification system is basically consistent with that in the amplification tube for nucleic acid collection and detection, the plasmid with the concentration of 1ng per microliter is added, and the primer sequence is replaced.

TABLE 2P 72 sequence

When starting the nucleic acid detection procedure, the swab is first sampled at the nasopharynx and placed in a viral sampling tube (VTM). After the swab is broken, the liquid of the sampling tube is injected into the sample lysate hole, and then an aluminum film is attached to start nucleic acid extraction. The magnetic bead collection well is now aligned with the sample lysis zone, i.e. the device is in the first position (lysis position).

And then vibrating, on one hand, the lysate is enabled to fully lyse viruses, so that the viruses release internal nucleic acids. The other side is oscillated to disperse the magnetic beads and combine with the nucleic acid in the sample. Shaking for about 30 seconds. And then the upper chip and the lower chip are put back on the base with the magnet, so that the magnetic beads are enriched, and the magnetic beads are sucked into the magnetic bead collecting holes below the sample lysate area.

The second step is a washing step. The upper chip is rotated counterclockwise on the base such that the cleaning liquid hole area of the upper chip is aligned with the magnetic bead collection hole, i.e., the second position (cleaning position). And then vibrating to enable the magnetic beads to be fully dispersed, so that the cleaning liquid in the reagent is washed away to be combined with the proteins on the magnetic beads, and the specificity of the whole extraction is ensured. Shaking for about 10 seconds. And then the upper chip and the lower chip are put back on the instrument base with the magnet, so that the magnetic beads are enriched and attracted to the magnetic bead collecting holes below the protein cleaning area.

The third step is the elution step. The upper chip is rotated counter-clockwise on the base such that the upper chip nucleic acid eluent hole is aligned with the magnetic bead collection hole, i.e., the third position (elution position). And then vibrating to enable the magnetic beads to be fully dispersed, so that nucleic acid in the magnetic beads is fully eluted, and meanwhile, liquid in the control reagent hole falls into the control reagent amplification tube below. Shaking for about 20 seconds. And then the upper chip and the lower chip are put back on the instrument base with the magnet, so that the magnetic beads are enriched and attracted to the magnetic bead collection hole below the nucleic acid eluting region.

At this time, the upper chip cannot rotate counterclockwise again through the deconstructed design.

Fourth step sample collection. The upper chip is rotated clockwise on the base such that the upper chip nucleic acid eluent aperture is aligned with the purified nucleic acid passing aperture. Depending on the design, it rotates counter-clockwise to the extreme position, i.e. the fourth position (elution position). And then vibrating for about 15 seconds, and fully dropping the liquid of the nucleic acid eluent into the amplification tube for collecting and detecting the nucleic acid below.

Thus, the whole process takes no more than 2 minutes after the nucleic acid is extracted by a simple manual magnetic bead method.

At this time, through structural design, the upper chip can not rotate clockwise any more. After amplification, the sample is subjected to nucleic acid amplification by a heating module in the base.

After 30 minutes, amplification is finished, and the LAMP reaction brings about pH change, so that the reaction system can be characterized by adopting a visual detection scheme through adding an indicator. One detection scheme is to present the results by visual detection. The user can directly observe through naked eyes without depending on intelligent equipment such as mobile phones, computers and the like, and the process of judging the result is omitted.

Through testing, the whole set of nucleic acid detection system has good experimental results based on at least the following properties:

1. the reproducibility of nucleic acid extraction using the present system is good. The nucleic acid is extracted by adopting a magnetic bead method, so that the extraction result has strong repeatability relative to the temperature. According to the experimental results, 3 times of extraction of the same sample are shown, and RT-PCR reaction is carried out on a standard PCR instrument by taking the sample as a template, wherein the CT value difference is within 0.5 (the specific result is shown in FIG. 29), and the sample has very good repeatability.

2. The magnetic bead method for extracting nucleic acid has higher extraction efficiency. Comparing the nucleic acid sample extracted by the method with the result of the nucleic acid sample extracted by the standard process, wherein the nucleic acid extracted by the standard process is strictly carried out according to the specification, and the whole process takes about 30 minutes after repeated shaking and centrifugation of professional equipment. The results show that the difference of CT values is still within 0.5, and the difference is very small. Meanwhile, the whole extraction result shows very good linearity, and the extraction effect of each time is proved to be relatively stable, so that the method has very strong practicability.

3. The sensitivity of nucleic acid extraction is higher than the prior art level. Currently, the sensitivity of nucleic acid detection is a very important factor. The national standard requires a detection sensitivity of 300 copies per milliliter. The invention can realize the detection sensitivity of 250 copies in 1mL sample, and basically meets the national standard.

Example 2

For the purpose of experimental detection with higher precision, the invention provides another scheme. According to the invention, as the CRISPR cas12 is added by a two-step method, more accurate detection of amplification results is realized, and false positives generated by other amplification results are avoided.

The upper chip design is shown in figure 30, and comprises a lysate hole, a cleaning solution hole, an eluent hole and a detection reagent hole.

An amplification tube is arranged under the purified nucleic acid passing hole only under the lower chip, and an amplification tube is not arranged under the contrast agent passing hole. The reagent pre-buried in the amplification tube can be a CRISPR cas12 reaction system, and the target sample can be detected more accurately through the specific recognition of CRISPR cas12 enzyme, so that false positives of detection results are avoided, and the detection sensitivity is improved. The detection result can be then used in combination with a fluorescent detection scheme.

4. Mu.L of 10 Xbuffer reagent, 1. Mu.M fluorescent probe, 500nM crRNA and 1. Mu.M Cas12a nuclease were added to the detection reagent wells of the upper chip. At this time, the reagent is contained in the upper chip.

After the first three steps (i.e., lysis, washing, elution) as described in example 1 are completed, the fourth step is to rotate the upper chip clockwise so that the eluent wells are aligned with the purified nucleic acid passing wells of the lower chip and the nucleic acid solution flows into the amplification tube. Is mixed with a first amplification reagent in an amplification tube and subjected to a first nucleic acid amplification reaction under conditions.

Fifth step is to add detection reagent. At this time, the upper chip is rotated counterclockwise again on the base so that the upper chip is returned to its detection reagent hole aligned with the purified nucleic acid passing hole of the lower chip. At this time, the TOLO reagent, fluorescent probe, crRNA, cas12a nuclease mixed reagent of the detection reagent well of the upper chip falls into the amplification tube again. Mixing with the product of the first amplification reaction, and performing CRISPR detection reaction under certain conditions.

With the aid of the invention, the above embodiment provides a better solution for the situation that the amplification reagent and the detection reagent cannot coexist.

It should be noted that in the present patent application, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element. In the present patent application, if it is mentioned that an action is performed according to an element, it means that the action is performed at least according to the element, and two cases are included: the act is performed solely on the basis of the element and is performed on the basis of the element and other elements. Multiple, etc. expressions include 2, 2 times, 2, and 2 or more, 2 or more times, 2 or more.

This specification includes combinations of the various embodiments described herein. Separate references to "one embodiment" or a particular embodiment, etc., do not necessarily refer to the same embodiment; however, unless indicated as mutually exclusive or as would be apparent to one of skill in the art, the embodiments are not mutually exclusive. It should be noted that the term "or" is used in this specification in a non-exclusive sense unless the context clearly indicates otherwise or requires otherwise.

All documents mentioned in the present application are considered to be included in the disclosure of the present application in their entirety, so that they may be subject to modification if necessary. Further, it will be understood that various changes or modifications may be made to the present application by those skilled in the art after reading the foregoing disclosure of the present application, and such equivalents are intended to fall within the scope of the present application as claimed.

Claims

1. The palm nucleic acid detection device is characterized by comprising a first module and a second module which can rotate along the same axis, wherein the first module comprises at least one group of cavities, and the group of cavities at least comprises a cavity for a cracking reaction, a cavity for a cleaning reaction and a cavity for an eluting reaction; the second module comprises a magnetic bead collecting hole for accommodating the magnetic beads and a nucleic acid collecting and detecting cavity; when the two modules rotate around the axle center relatively, the palm nucleic acid detection device at least forms the following steps:

2. The palm nucleic acid detecting apparatus according to claim 1, wherein the magnetic beads are dispersed into the cavity of the first module containing the solution under the action of the external force at the first position and/or the second position and/or the third position, and then fall into the magnetic bead collection hole of the second module again.

3. The palm nucleic acid detecting device of claim 1, wherein the set of cavities of the first module further comprises a cavity for receiving a control reagent.

4. The palm nucleic acid detecting device according to claim 1, wherein the volume of the magnetic bead collection well is 10% or less of the volume of the cavity for elution reaction.

5. The palm nucleic acid detecting apparatus according to claim 1, wherein the external force for moving the magnetic beads up and down between the first module and the second module is any one or a combination of magnetic force and gravity.

6. The palm nucleic acid detecting device of claim 2, wherein the nucleic acid collection detection chamber of the second module is an amplification tube.

7. The palm nucleic acid detecting device according to claim 6, wherein the nucleic acid collecting and detecting chamber includes a nucleic acid detecting reagent selected from any one of: variable temperature amplification reagents, isothermal amplification reagents, hybridization-based nucleic acid detection reagents.

8. The palm nucleic acid detecting apparatus according to claim 1 or 3, wherein the palm nucleic acid detecting apparatus comprises a plurality of cavity groups, and the second module comprises a plurality of magnetic bead collection wells and a plurality of nucleic acid collection detection cavities corresponding to the first module.

9. A palm nucleic acid detection system comprising the palm nucleic acid detection device according to any one of claims 1 to 8, further comprising: the base, the base is a sealed housing of uncapping type, and inside cavity is provided with and is equipped with the buckle with palm nucleic acid detecting device adaptation in order to fix it, the base includes: control module, heating module, magnetic force module, wherein

10. The palm nucleic acid detection system of claim 9, further comprising an optical detection module configured to illuminate with excitation light to cause fluorescence of the substance within the nucleic acid collection detection chamber and to record the resulting fluorescence signal.

11. The palm nucleic acid detection system of claim 9 or 10, further comprising a data transmission module including at least the capability to communicate wirelessly over short distances with a consumer electronic device including any one or more of a smart phone, a personal computer, a smart watch, and a tablet computer.

12. A palm nucleic acid detection method, comprising the steps of:

(S1) taking the palm nucleic acid detecting system according to any one of claims 9 to 11, wherein a lysis solution, a cleaning solution and an eluent are respectively preset in the cavity for the lysis reaction, the cavity for the cleaning reaction and the cavity for the elution reaction of the first module; a nucleic acid detection reagent is preset in the nucleic acid collection detection cavity of the second module; after tearing the membrane, adding a sample into the cavity for the cleavage reaction;