CN214361367U

CN214361367U - Nucleic acid detection device

Info

Publication number: CN214361367U
Application number: CN202023198969.4U
Authority: CN
Inventors: 牛小牧; 陆军
Original assignee: Shandong Shunfeng Biotechnology Co Ltd
Current assignee: Shandong Shunfeng Biotechnology Co Ltd
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2021-10-08
Anticipated expiration: 2030-12-25

Abstract

The utility model discloses a nucleic acid detection device, including the reagent pipe, can dismantle the detecting tube of being connected and set up in the test paper of detecting tube with the reagent pipe, reagent pipe and detecting tube enclose into the reaction chamber that holds test paper, and the reagent pipe forms the water conservancy diversion passageway in reaction tank, medicament groove and intercommunication reaction tank and medicament groove, and the water conservancy diversion passageway forms the throat with medicament groove intercommunication department. The reaction tank is communicated with the reagent tank through the diversion channel, when the reagent pipe inclines, a second reagent in the reagent tank flows into the diversion channel through the necking, and the second reagent flows in the diversion channel in a dripping shape to enter a sample to be detected in the reaction tank, so that the reaction of the reagent and the sample to be detected is more sufficient.

Description

Nucleic acid detection device

Technical Field

The utility model relates to the field of biotechnology, concretely relates to nucleic acid detection device.

Background

After the new coronavirus infects human body, it will propagate in respiratory tract system, so it can judge whether human body is infected by virus by detecting virus nucleic acid in sputum and nasopharyngeal swab. Nucleic acid detection plays a key role in the diagnosis of new coronavirus infection. In hospitals, nucleic acid detection is the gold standard for diagnosing new coronavirus infections. Outside hospitals, nucleic acid detection can also be used for large-scale screening to find out asymptomatic infectors who are still latent as much as possible. The risk of population infection can be greatly reduced by screening, and the detection of the novel coronas is a detection means which has wide application range and is easy to obtain.

The principle of loop-mediated isothermal amplification (LAMP) is that 4 primers are used to identify 6 specific regions on a target gene, and strand displacement type DNA polymerase is used to amplify the target gene at a constant temperature of 60-65 ℃, so that a large amount of magnesium pyrophosphate white precipitate is generated in the reaction, and the color change can be observed directly by naked eyes or by adding a fluorescent dye. The technology can be comparable to or even superior to Polymerase Chain Reaction (PCR) technology in the indexes such as sensitivity, specificity, detection range and the like, does not depend on any special instrument and equipment to realize on-site high-flux rapid detection, and has detection cost far lower than that of fluorescent quantitative PCR. The method reduces the influence caused by temperature rise and drop of the polymerase chain reaction technology and the requirements on expensive and precise experimental instruments, and has high amplification efficiency.

The on-site timely detection of nucleic acid has a good application scene to epidemic situation prevention and control and epidemiological investigation, but the detection device of present on-site instant detection can't guarantee that reagent is not polluted by the sample that awaits measuring, and simultaneously, when adopting detection device to carry out liquid medicine and the sample that awaits measuring and mixing, the interpolation effect that can't guarantee the liquid medicine is accomplished unanimously with manual operation, no matter the liquid medicine adds too slowly or adds to the sample department that awaits measuring in a strand, can all cause the influence to the reaction of liquid medicine and sample that awaits measuring, and then influence the testing result.

SUMMERY OF THE UTILITY MODEL

To the above-mentioned not enough, the utility model provides a nucleic acid detecting device, this nucleic acid detecting device can control the liquid medicine and add the speed to the sample that awaits measuring in.

The utility model discloses a realize through following technical scheme:

the utility model provides a nucleic acid detection device, includes the reagent pipe, can dismantle the detection tube of being connected and set up in the test paper of detection tube with the reagent pipe, and reagent pipe and detection tube enclose into the reaction chamber that holds test paper, and the reagent pipe forms reaction tank, medicament groove and the water conservancy diversion passageway of intercommunication reaction tank and medicament groove, and water conservancy diversion passageway and medicament groove intercommunication department form the throat. The reaction tank is communicated with the reagent tank through the diversion channel, when the reagent pipe inclines, a second reagent in the reagent tank flows into the diversion channel through the necking, and the second reagent flows in the diversion channel in a dripping shape to enter a sample to be detected in the reaction tank, so that the reaction of the reagent and the sample to be detected is more sufficient.

Furthermore, a flaring is formed at the communication position of the flow guide channel and the reaction tank. The second reagent in the reagent groove flows into the diversion channel in a dripping shape through the necking and then is gathered through the flaring, and the dripping reagent can quickly flow into the reaction groove from the flaring when flowing in the diversion channel, so that the reaction of the second reagent and the sample in the reaction groove is accelerated.

Further, the flow guide channel is of a pyramid-shaped structure. The flow guide channel is communicated with the reaction groove and the reagent groove in a linear structure, so that the flow path of the second reagent is more accurate, and the second reagent can flow into the reaction groove.

Furthermore, the reagent tube is sunken to form a buffer cavity, and the reaction tank and the reagent tank are opened on the bottom wall of the buffer cavity. The reagent tube and the detection tube are detachably connected, and a buffer cavity is formed by the reagent tube in a concave manner between the reagent tube and the detection tube. A buffer area is reserved for the detection test paper in the detection tube and the reagent groove and the reaction groove in the reagent tube through the buffer cavity, so that the detection test paper can be prevented from being in contact reaction with the reagent or the sample in advance, and the detection result is prevented from being influenced.

Further, the flow guide channel is formed by sinking the bottom wall. The opening in reaction tank and medicament groove is located the diapire of cushion chamber, and water conservancy diversion passageway connection reaction tank and medicament groove lie in the cushion chamber diapire simultaneously, and water conservancy diversion passageway and diapire parallel and level are sunken to be formed by the diapire, have guaranteed that the medicament is stable gentle when water conservancy diversion passageway flows.

Further, the flow guide channel extends from the reagent tank toward the reaction tank in an inclined manner. The diversion channel is inclined and extended to ensure that the reagent flows into the reaction tank from the reagent tank, so that the sample to be detected is prevented from flowing back to pollute the second reagent, and the second reagent is ensured to flow into the reaction cavity rapidly and smoothly for reaction.

Further, the reaction groove forms a reaction groove bulge positioned at the lower part of the reagent tube, the reagent groove forms a reagent groove bulge positioned at the lower part of the reagent tube, and the height of the reaction groove bulge is greater than that of the reagent groove bulge. The medicament groove bulge and the reaction groove bulge are formed on the upper part of the reagent tube, and the height of the bulge of the reaction groove is greater than that of the medicament groove, so that isothermal amplification reaction can be conveniently carried out on the medicament groove by heating. Meanwhile, the reaction tank and the reagent tank can be distinguished through the difference of the heights of the protrusions, and the storage positions of the reagents and the samples can be known after the reagent tube and the detection tube are connected and sealed. In addition, the higher arch of reaction tank can slow down the evaporation rate of sample when heating, guarantees the accuracy of testing result.

Further, the inner diameter of the reaction tank is smaller than that of the medicament tank. Since the medicament groove is formed to have a low protrusion, the volume of the reagent can be increased by appropriately increasing the inner diameter of the medicament groove.

Further, the reaction groove bulge is sequentially provided with a cylindrical section and a conical section which are connected with the reagent tube along the extending direction of the reaction groove bulge. Through setting up the reaction tank into cylindricality section and toper section, enlarged the heating area of tube seat, be convenient for heat the reaction tank and make it be heated evenly, the reaction is more abundant.

Further, the detection tube is made of transparent material. The reagent tube and the detection tube are connected to form a closed space, the detection tube is made of transparent materials, the result of the reaction of the detection test paper, the reagent and the sample can be visually displayed, and the reagent tube and the detection tube are convenient and visual.

Drawings

FIG. 1 is a front view showing an exemplary embodiment of a nucleic acid detecting apparatus according to the present invention;

FIG. 2 is a schematic view illustrating a state of use of the nucleic acid detecting device according to the present invention;

FIG. 3 is a block diagram illustrating an exemplary embodiment of a reagent vessel according to the present invention;

fig. 4 is a diagram illustrating an exemplary embodiment of a detection tube according to the present invention.

Reference numerals:

1. reagent pipe, 11, reagent groove, 12, reaction tank, 121, cylindrical section, 122, conical section, 13, diversion channel, 131, throat, 132, flaring, 14, buffer cavity, 15, bottom wall, 2, detection tube, 21, mounting groove.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by a person skilled in the art without creative work belong to the protection scope of the present invention.

It will be understood that when an element is referred to as being "on" or "disposed of" another element, it can be on the other element or intervening elements may also be present. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or intervening elements may also be present.

As shown in FIGS. 1 to 3, a nucleic acid detecting device comprises a reagent tube 1, a detecting tube 2 detachably connected with the reagent tube 1, and a detecting test paper arranged on the detecting tube 2, wherein the reagent tube 1 and the detecting tube 2 enclose a reaction cavity for accommodating the detecting test paper, the reagent tube 1 forms a reaction tank 12, a reagent tank 11, and a flow guide channel 13 for communicating the reaction tank 12 with the reagent tank 11, and a necking 131 is formed at the communication position of the flow guide channel 13 and the reagent tank 11. The sample to be detected is added into the reaction tank 12 in the reagent tube 1 and reacts with the first reagent preset in the reaction tank 12, the reaction tank 12 is communicated with the reagent tank 11 through the diversion channel 13, when the reagent tube 1 is inclined, the second reagent in the reagent tank 11 flows into the diversion channel 13 through the necking 131, and the second reagent flows into the sample to be detected in the reaction tank 12 in a dripping shape in the diversion channel 13, so that the reaction of the reagent and the sample to be detected is more sufficient.

Because the reaction between the reagent and the sample to be detected and the detection of the detection test paper are realized in the closed reaction cavity, the detection tube 2 is preferably made of transparent material in order to completely reduce the possibility of external pollution and interference; the condition of the test paper can be observed without disassembling the detection tube 2 and the reagent tube 1, so that external pollution is avoided, and full-closed detection is realized.

One specific embodiment is provided below to illustrate the method of using the detection device of the present application.

The reaction tank is pre-loaded with a first reagent, preferably, a PCR reaction reagent, for example, a LAMP reaction reagent.

The agent groove is preset with a second reagent, and the second reagent comprises a Cas protein with trans cleavage activity, a gRNA and a single-stranded nucleic acid detector; the gRNA includes a region that binds to the Cas protein described above and a region that hybridizes to a target nucleic acid to be detected.

The detection test paper is a colloidal gold detection test paper.

In one embodiment, a reaction tank is preset with a first reagent, a sample to be tested is added into the reaction tank, and the sample to be tested is mixed with the first reagent and reacts at a first temperature; for example, a LAMP reaction reagent is provided in the reaction tank, and the nucleic acid in the sample to be tested can be amplified at a temperature suitable for the LAMP reaction (for example, 60 to 65 ℃).

The reagent groove is preset with a second reagent; in one embodiment, the second agent comprises a Cas protein (alternatively referred to as a CRISPR protein) having trans cleavage activity, e.g., Cas12a, Cas12b, Cas12i, Cas12j, Cas13a, and the like; the second reagent also comprises a gRNA which comprises a region combined with the Cas protein and a region hybridized with a target nucleic acid to be detected; in addition, the second reagent further comprises a single-stranded nucleic acid detector.

Amplifying to obtain nucleic acid in the sample after the reaction in the reaction tank is carried out for a period of time; the second reagent in the reagent reservoir is added to the reaction reservoir and the reaction is continued for a period of time at a temperature suitable for reaction of the Cas protein. Under the reaction condition, if the target nucleic acid to be detected exists in the sample to be detected, the Cas protein in the system cuts the single-stranded nucleic acid detector; if the target nucleic acid to be detected does not exist in the sample to be detected, the Cas protein in the system will not cut the single-stranded nucleic acid detector.

After the reaction, the nucleic acid detecting device is turned over so that the reaction liquid flows to the detection groove and contacts with the detection test paper for detection.

The arrangement of the single-stranded nucleic acid detector and the detection test paper can refer to Chinese patent application CN 111996236A; specifically, the method comprises the following steps: the single-stranded nucleic acid detector has a first molecule (e.g., FAM or FITC) attached to the 5 'end and a second molecule (e.g., biotin) attached to the 3' end. The reaction system containing the single-stranded nucleic acid detector is matched with a detection test paper to detect the target nucleic acid (preferably, a colloidal gold detection mode). The test strip is designed with two capture lines, with an antibody that binds to a first molecule (i.e., a first molecular antibody) at the sample contacting end (colloidal gold), an antibody that binds to the first molecular antibody at the first line (colline), and an antibody that binds to a second molecule (i.e., a second molecular antibody, such as avidin) at the second line (testline). As the reaction flows along the strip, the first molecular antibody binds to the first molecule carrying the cleaved or uncleaved single-chain nucleic acid detector to the capture line, the cleaved reporter will bind to the antibody of the first molecular antibody at the first capture line, and the uncleaved reporter will bind to the second molecular antibody at the second capture line. Binding of the reporter group at each line will result in a strong readout/signal (e.g. color). As more reporters are cut, more signal will accumulate at the first capture line and less signal will appear at the second line. According to the reaction result of the test paper, whether the target nucleic acid to be detected exists in the sample can be reflected. In certain aspects, the molecules in the single stranded nucleic acid detector can be substituted for each other, or the position of the molecules can be altered.

According to the reaction result of the test paper, whether the target nucleic acid to be detected exists in the sample to be detected can be judged.

Specifically, the sample to be tested may be derived from viruses, bacteria, microorganisms, soil, water sources, human bodies, animals, plants. In the present application, the sample to be tested is a biological sample; it is understood that the biological sample may be a plant cell, callus, tissue or organ (e.g., root, stem, leaf, flower, seed, fruit), and the like.

Wherein a biological sample is any solid or fluid sample obtained, excreted or secreted from any organism, including but not limited to unicellular organisms such as bacteria, yeasts, protozoa and amoebae and the like, multicellular organisms (e.g. plants or animals, including samples from healthy or superficially healthy human subjects or human patients affected by a condition or disease to be diagnosed or investigated, e.g. infection by a pathogenic microorganism such as a pathogenic bacterium or virus).

For example, the biological sample may be a biological fluid obtained from, for example, blood, plasma, serum, urine, feces, sputum, mucus, lymph, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, exudate (e.g., obtained from an abscess or any other site of infection or inflammation), or a fluid obtained from a joint (e.g., a normal joint or a joint affected by a disease, such as rheumatoid arthritis, osteoarthritis, gout, or septic arthritis), or a swab of a skin or mucosal surface. The sample may also be a sample obtained from any organ or tissue (including biopsies or autopsy specimens, e.g., tumor biopsies) or may comprise cells (primary cells or cultured cells) or culture medium conditioned by any cell, tissue or organ. Exemplary samples include, but are not limited to, cells, cell lysates, blood smears, cytocentrifuge preparations, cytological smears, bodily fluids (e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.), tissue biopsies (e.g., tumor biopsies), fine needle aspirates, and/or tissue sections (e.g., cryostat tissue sections and/or paraffin-embedded tissue sections).

Referring to fig. 1 and 2, the reaction tank 12 is formed with a reaction tank protrusion located at a lower portion of the reagent tube 1, and the reagent tank 11 is formed with a reagent tank protrusion located at a lower portion of the reagent tube 1, and the height of the reaction tank protrusion is greater than that of the reagent tank protrusion. The reagent groove protrusion and the reaction groove protrusion are both formed on the upper part of the reagent tube 1, and the protrusion height of the reaction groove 12 is larger than that of the reagent groove 11, so that the reagent groove 11 can be heated to perform isothermal amplification reaction. Meanwhile, the reaction tank 12 and the reagent tank 11 can be distinguished through the difference of the heights of the protrusions, and the storage positions of the reagents and the samples can be known after the reagent tube 1 and the detection tube 2 are connected and sealed. In addition, the higher projection of the reaction tank 12 can slow down the evaporation speed of the sample during heating, and the accuracy of the detection result is ensured.

The reaction groove bulge is sequentially provided with a cylindrical section 121 and a conical section 122 which are connected with the reagent tube 1 along the extension direction of the reaction groove bulge; the cylindricality section 121 is arranged in increasing the volume that holds of reaction tank 12, and toper section 122 is convenient for the reaction tank arch and is inserted and be used for heating the instrument, and reagent pipe 1 is fixed effectual when the heating, simultaneously, because the bellied height in reaction tank is greater than the bellied height in medicament groove, when heating the reaction tank arch, also can reduce the influence of heating instrument to second reagent in the medicament groove 11.

It should be noted that the cylindrical section 121 may be a cylindrical section 121, and may also be a prismatic section 121.

As shown in FIG. 3, the reagent vessel 1 is recessed to form a buffer chamber 14, and the reaction tank 12 and the reagent tank 11 are opened at a bottom wall 15 of the buffer chamber 14; the reagent tube 1 and the detection tube 2 are detachably connected, when the nucleic acid detection device is used, a collected sample to be detected is placed in the reaction tank 12, a second reagent is placed in the reagent tank 11, the reagent tube 1 and the detection tube 2 are connected to form a closed space, the buffer cavity 14 formed by sinking the reagent tube 1 in the middle of the reagent tube 1 and the detection tube 2 forms a three-cavity design of the nucleic acid detection device, and different reactions of two tube cavity parts can be buffered and isolated. Buffer areas are reserved for the test paper in the detection tube 2 and the medicament groove 11 and the reaction groove 12 in the reagent tube 1 through the buffer cavity 14, so that the test paper can be prevented from contacting and reacting with the reagent or the sample in advance, and the detection result is prevented from being influenced.

The flow guide channel 13 is formed by recessing the bottom wall 15. The nucleic acid detecting apparatus is designed to react a detection reagent with a sample to be detected to obtain a detection result. The openings of the reaction tank 12 and the reagent tank 11 are located on the bottom wall 15 of the buffer cavity 14, the guide channel 13 is connected with the reaction tank 12 and the reagent tank 11 and is located on the bottom wall 15 of the buffer cavity 14, the guide channel 13 is flush with the bottom wall 15 and is formed by sinking the bottom wall 15, and stability and smoothness of the second reagent when the guide channel 13 flows are guaranteed.

The communication part of the flow guide channel 13 and the reaction tank 12 forms a flaring 132. The second reagent in the reagent tank 11 flows into the flow guide channel 13 in a drop shape through the necking 131 and then is converged through the flaring 132, and the drop-shaped reagent can flow into the reaction tank 12 from the flaring 132 when flowing in the flow guide channel 13, so that the reaction between the second reagent and the sample in the reaction tank 12 is accelerated.

The flow guide channel 13 is of a pyramid-shaped structure. For the nucleic acid detecting device, the flow guide channel 13 is set to be a pyramid-shaped structure, so as to ensure that the reaction tank 12 and the reagent tank 11 are communicated, so that the second reagent in the reagent tank 11 can flow into the reaction tank 12 to react with the sample to be detected. The accuracy of the flow path of the second reagent is ensured, and the second reagent can flow into the reaction tank 12, wherein the bottom end of the flow guide channel 13 is the flared opening 132, and the top end of the flow guide channel 13 is the necking opening 131.

The inner diameter of the reaction tank 12 is smaller than the inner diameter of the chemical tank 11. Since the medicament groove is formed to have a lower projection, the capacity of the reagent can be increased by appropriately increasing the inner diameter of the medicament groove 11. And by appropriately reducing the inner diameter of the reaction tank 12, the cross-sectional area of the reaction tank 12 can be reduced to slow down the evaporation rate of the sample.

The guide passage 13 extends obliquely from the chemical tank 11 toward the reaction tank 12. The inclined extension of the diversion channel 13 ensures that the reagent flows into the reaction tank 12 from the reagent tank 11, so as to prevent the sample to be detected from flowing back to pollute the second reagent and ensure that the second reagent quickly and smoothly flows into the reaction cavity for reaction.

As shown in fig. 1, the reagent vessel 1 is formed with a threaded portion; set up the screw thread in reagent pipe 1 department, when can guaranteeing that reagent pipe 1 and test tube 2 are connected, sealing performance is better, because, reagent intraductal adding first reagent, second reagent and the sample that awaits measuring, consequently, when the installation, need install the test tube to reagent pipe department, consequently, screw thread portion is formed in reagent pipe 1, makes things convenient for the test tube to aim at the installation, and wherein, screw thread portion is formed in the outside of reagent pipe 1.

The detecting tube is when being connected with the reagent pipe, the detecting tube is formed with the smooth portion with screw portion complex, though there is screw portion, but reagent pipe 1 and detecting tube 2 do not pass through threaded connection, avoid many times rotatory increase operating time, and reagent pipe 1 is when being connected with detecting tube 2 lock, screw portion can increase the friction between reagent pipe 1 and detecting tube 2 again, not only prevent to slide between reagent pipe 1 and detecting tube 2, still increased the leakproofness between reagent pipe 1 and detecting tube 2, prevent that reagent and sample are contaminated, avoid the testing result inaccurate.

Also, referring to fig. 4, preferably, a screw portion is also formed at the sensing tube 2, and the mounting groove 21 is located at a lower portion of the screw portion.

It will be appreciated that the threaded portion may be any raised structure that provides a sealing function, such as an annular rib, to ensure sealing.

Preferably, the test tube 2 is including setting up test paper's mounting groove 21, fix test paper through mounting groove 21, prevent that test paper from rocking in test tube 2, the influence with wait to detect the liquid contact, it is concrete, mounting groove 21 includes two "U" shape posts, test paper is located test tube 2's middle zone, the opening direction of two "U" shape posts all faces the axis department of test tube 2 promptly, test paper can contact and detect with the solution after reagent and the sample mixture rapidly, the efficiency of detection is improved, shorten the time that solution flow direction detected test paper simultaneously, the testing result is more accurate.

Or, the mounting groove includes two "L" shape posts, and the first panel of two "L" shape posts is parallel and meets with the inner wall of detecting tube 2, and the second panel of two "L" shape posts is located the coplanar and leaves the space between the second panel of two "L" shape posts, is convenient for observe the installation condition of test paper from the space between, and it can be understood that, also can be closely laminated between the second panel of two "L" shape posts.

The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. The nucleic acid detection device is characterized by comprising a reagent tube, a detection tube detachably connected with the reagent tube and detection test paper arranged on the detection tube, wherein the reagent tube and the detection tube enclose a reaction cavity for accommodating the detection test paper, the reagent tube forms a reaction groove, a reagent groove and a flow guide channel for communicating the reaction groove and the reagent groove, and a necking is formed at the communication position of the flow guide channel and the reagent groove.

2. The nucleic acid detecting apparatus according to claim 1, wherein a flare is formed at a position where the flow guide channel communicates with the reaction chamber.

3. The nucleic acid detecting device according to claim 1, wherein the flow guide channel has a pyramid-like structure.

4. The nucleic acid detecting device according to claim 1, wherein the reagent tube is recessed to form a buffer chamber, and the reaction well and the reagent well are opened to a bottom wall of the buffer chamber.

5. The nucleic acid detecting device according to claim 4, wherein the flow guide channel is formed by recessing the bottom wall.

6. The nucleic acid detecting apparatus according to claim 5, wherein the flow guide channel extends obliquely from the reagent reservoir toward the reaction reservoir.

7. The nucleic acid detecting apparatus according to claim 1, wherein the reaction well forms a reaction well protrusion located at a lower portion of the reagent tube, the reagent well forms a reagent well protrusion located at a lower portion of the reagent tube, and a height of the reaction well protrusion is larger than a height of the reagent well protrusion.

8. The nucleic acid detecting apparatus according to claim 7, wherein an inner diameter of the reaction tank is smaller than an inner diameter of the reagent tank.

9. The apparatus according to claim 7, wherein the reaction well protrusion has a cylindrical section and a tapered section connected to the reagent tube in this order along the extension direction.

10. The nucleic acid detecting apparatus according to claim 1, wherein the detecting tube is made of a transparent material.