Fully automatic nucleic acid extraction, amplification and detection integrated reaction tube and columnar detection card

Abstract

The present invention relates to the field of in vitro diagnostic nucleic acid detection, and provides a fully automatic integrated reaction tube for nucleic acid extraction, amplification and detection and a columnar detection card. Among them, along the direction from the mouth to the bottom, this integrated reaction tube successively includes a right-angled frustum section, a cylindrical section and a right-angled cone section. The right-angled frustum section constitutes the lysis area, the cylindrical section constitutes the washing area, and the right-angled cone section constitutes the elution and amplification area. The right-angled side of the right-angled frustum section and the right-angled side of the right-angled cone section are on the same side and flush with the side of the cylindrical section. This columnar detection card is the above-mentioned integrated reaction tube filled with lysis solution, washing solution and reaction solution, and the various liquids are separated by phase change separators. This reaction tube can not only complete the entire extraction, amplification and detection process in one tube, but also has the characteristics of simple structure, convenient production, and simple magnetic movement structure of the supporting detection equipment.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic nucleic acid detection, and particularly to a fully automatic nucleic acid extraction, amplification, and detection integrated reaction tube, and a columnar detection card using the reaction tube. Background Art

[0002] From nucleic acid extraction to amplification and detection, it includes steps such as lysis, washing, elution, and amplification. Currently, the mainstream technology must be carried out step by step in a standard laboratory. The whole process is prone to contaminating the sample nucleic acid, with high operation difficulty and high requirements for operators, which is not conducive to the popularization of nucleic acid detection technology.

[0003] The Chinese patent application for invention with the publication number CN111876468A discloses a fully automatic nucleic acid detection test tube, which includes: a main tube body, an annular tube groove, a conical tube body, and a flip cover; the main tube body, the annular tube groove, and the conical tube body are connected through the side; the main tube body is the lysis solution area; the annular tube groove is the cleaning area; the conical tube is the reaction area. Its detection principle is: an external magnet is arranged at the communication port of the main tube body and the annular tube groove. When the sample is lysed in the main tube body, the magnetic beads gather under the attraction of the external magnet. The external magnet rotates around the side wall of the annular tube groove for one week, driving the magnetic beads to pass through the hydrophobic substance from the main tube body, washing away the impurities of the magnetic beads with the cleaning solution in the annular tube groove, and passing through the hydrophobic substance into the conical tube body for nucleic acid reaction.

[0004] In the above-mentioned patent application for invention, the lysis solution tube, the cleaning solution tube, and the reaction solution tube are connected together, and the whole extraction and amplification process is carried out under closed conditions, effectively avoiding environmental pollution. Moreover, the whole experimental process automatically completes the extraction, amplification, and detection through equipment, greatly simplifying the experimental operation difficulty. However, there are still the following defects: 1. The structure of the detection test tube is complex, which is not conducive to production. Since the detection test tube includes an annular tube groove, the structure is complex, so it is not conducive to the production of the detection test tube and the packaging of the three liquids. 2. The movement directions of the external magnets are inconsistent, which will increase the complexity of the supporting detection equipment. The movement track of the magnetic beads is: first move downward in the main tube body into the annular tube groove, rotate around the annular tube groove for one week, and then move downward into the conical tube body. The movement of the magnetic beads is driven by the external magnet, so the external magnet needs to be able to move up and down and also be able to make a circular motion around the annular tube groove, which makes the structure of the supporting detection equipment complex. Summary of the Invention

[0005] The purpose of the present invention is to provide a fully automatic nucleic acid extraction, amplification, and detection integrated reaction tube to at least solve the above-mentioned defects existing in the related technology to a certain extent.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A fully automatic integrated reaction tube for nucleic acid extraction, amplification and detection. Along the direction from the mouth to the bottom, the integrated reaction tube sequentially includes a right-angled frustum section, a cylindrical section and a right-angled cone section. The right-angled frustum section constitutes the lysis zone, the cylindrical section constitutes the washing zone, and the right-angled cone section constitutes the elution and amplification zone. The right-angled side of the right-angled frustum section and the right-angled side of the right-angled cone section are on the same side and flush with the side of the cylindrical section.

[0008] In the above-mentioned fully automatic integrated reaction tube for nucleic acid extraction, amplification and detection, preferably, the right-angled frustum section, the right-angled cone section and the cylindrical section are respectively provided with a heating structure on the side opposite to the right-angled side, and during the detection process, the heating structure can be fitted with the heat conduction surface of an external heating body. More preferably, the heating structure is an arc surface adapted to the reaction tube.

[0009] In the above-mentioned fully automatic integrated reaction tube for nucleic acid extraction, amplification and detection, preferably, the inner diameter of the cylindrical section is smaller than the average inner diameter of the right-angled frustum section and larger than the average inner diameter of the right-angled cone section. More preferably, the inner diameter of the cylindrical section is 2 to 10 mm, and the ratio of the length to the inner diameter of the cylindrical section is greater than 5.

[0010] In the above-mentioned fully automatic integrated reaction tube for nucleic acid extraction, amplification and detection, preferably, the bottom of the integrated reaction tube includes a semi-spherical structure, and the mouth of the integrated reaction tube includes a flip cover integrally formed with the integrated reaction tube.

[0011] A columnar detection card, which includes a lysis solution, a washing solution, a reaction solution, a phase change separator and a reaction tube; the reaction tube is the integrated reaction tube described in any one of the above; the lysis solution, the washing solution and the reaction solution are respectively contained in the lysis zone, the washing zone and the elution and amplification zone of the integrated reaction tube, and phase change separators are respectively arranged between the lysis solution and the washing solution, and between the washing solution and the reaction solution. The mouth of the integrated reaction tube is sealed by an integrally formed flip cover. At the storage temperature, the phase change separator is in a solid state, and at the reaction temperature, the phase change separator turns into a liquid state.

[0012] Compared with the prior art, the present invention has at least the following beneficial effects:

[0013] The integrated reaction tube with a three-section structure is adopted, and there is no other structure inside the tube body, so the structure is simple and convenient for production. At the same time, during the experiment, the washing movement direction of the magnetic bead-nucleic acid complex is the same as its transmission direction between adjacent sections, which enables the external magnet driving the movement of the magnetic bead to move only in one direction (along the axis direction of the reaction tube). Therefore, it also has the characteristic of simple magnetic body movement structure of the required supporting detection equipment.

[0014] The right-angled frustum design and the right-angled cone design, on the one hand, enable the cracking area and the elution amplification area to form a single-sided contraction structure respectively, which is beneficial to increasing the capacity of the cracking area and saving the usage amount of the reaction solution. On the other hand, their right-angled sides are on the same side and flush with the side of the cylindrical section, so that during the entire transfer process of the magnetic beads, the suction force of the external magnet on the magnetic beads remains consistent, and magnetic bead loss can be avoided.

[0015] By using this integrated reaction tube, the entire extraction and amplification process is completed in one reaction tube, with simple operation, which can effectively avoid environmental pollution and can achieve fully automatic nucleic acid extraction, amplification, and detection.

[0016] The heating structure extends to the cracking area (right-angled frustum section), which can heat the cracking area and helps to crack some complex samples.

[0017] The specifications of its cylindrical section enable the magnetic bead-nucleic acid complex to have enough time to remove the residual lysis solution and sample on the surface, can reduce the usage amount of the washing solution, and can improve the stability of the entire filling liquid stratification system.

[0018] The semi-spherical structure at the bottom can condense the detection light, which is beneficial to improving the detection accuracy. Description of the Drawings

[0019] Figure 1 It is a schematic structural diagram of an embodiment of the integrated reaction tube;

[0020] Figure 2 It is a cross-sectional view thereof;

[0021] Reference numerals: 1, flip cover; 2, mouth part; 3, right-angled side; 4, semi-spherical structure; 5, right-angled frustum section; 6, cylindrical section; 7, right-angled cone section; 8, heating structure; 9, cracking area; 10, washing area; 11, elution amplification area. Detailed Embodiments

[0022] The present invention will be further described below with reference to the drawings and embodiments.

[0023] Embodiment 1:

[0024] This integrated reaction tube is a test tube used in in vitro diagnostic nucleic acid detection, and is a test tube integrating a cracking container, a washing container, an amplification reaction container, and a detection container.

[0025] Please refer to Figures 1 to 2, along the direction from the mouth 2 to the bottom, this integrated reaction tube successively includes a right-angled frustum section 5, a cylindrical section 6, and a right-angled conical section 7. Among them, the right-angled frustum section 5 constitutes the cracking zone 9, the cylindrical section 6 constitutes the washing zone 10, and the right-angled conical section 7 constitutes the elution amplification zone 11. And the right side 3 of the right-angled frustum section 5 and the right side 3 of the right-angled conical section 7 are on the same side ( Figure 1 in the left side in the figure), and are flush with the side of the cylindrical section 6. It can be seen that the left side of the tube body is a straight line.

[0026] Figure 1 In the figure, the right side of the right-angled frustum section 5, the right side of the right-angled conical section 7, and the right side of the cylindrical section 6, that is, the side opposite to the right side 3 ( Figure 1 in the left side in the figure), are provided with a heating structure 8. The heating structure 8 can be any surface structure that can fit the heat conduction surface of the external heating body, preferably an arc surface adapted to the reaction tube. This design enables the right side of the right-angled frustum section 5, the right side of the right-angled conical section 7, and the right side of the cylindrical section 6 to fit the heat conduction surface of the external heating body during the detection process, and efficiently conduct the heat energy of the external heating body to the corresponding reaction zone to precisely control the reaction temperature.

[0027] Furthermore, the inner diameter of the cylindrical section 6 is smaller than the average inner diameter of the right-angled frustum section 5 and larger than the average inner diameter of the right-angled conical section 7. The inner diameter of the cylindrical section 6 is preferably 2 to 10 mm, and the ratio of the length to the inner diameter of the cylindrical section 6 is greater than 5.

[0028] Furthermore, a semi-spherical structure 4 is further included at the bottom of the integrated reaction tube, and the mouth 2 of the integrated reaction tube further includes a flip cover 1 integrally formed with the integrated reaction tube.

[0029] Embodiment 2:

[0030] A columnar detection card based on the above reaction tube is used for in vitro diagnostic nucleic acid detection. This columnar detection card includes a lysis solution, a washing solution, a reaction solution, and the above integrated reaction tube; among them, the lysis solution, the washing solution, and the reaction solution are respectively contained in the cracking zone 9, the washing zone 10, and the elution amplification zone 11 of the integrated reaction tube. Phase change separators are respectively arranged between the lysis solution and the washing solution, and between the washing solution and the reaction solution. The mouth 2 of the integrated reaction tube is sealed with the flip cover 1. The phase change separator is a material that is in a solid state at the storage temperature and turns into a liquid state at the reaction temperature, preferably a material that is not miscible with the lysis solution, the washing solution, and the reaction solution, such as paraffin.

[0031] The preparation method of the columnar detection card is as follows: Load the reaction solution into the elution and amplification area 11 of the above integrated reaction tube, then load a layer of phase change separator, then load the washing solution into the washing area 10, and then load a layer of phase change separator above the washing solution. Then load the lysis solution into the lysis area 9, and seal the opening 2 with the flip cover 1 to form the columnar detection card.

[0032] The method for nucleic acid detection using the above columnar detection card is as follows:

[0033] Before the reaction, add the sample and magnetic beads to the lysis area 9, and after lysing for a period of time, the magnetic beads and nucleic acid bind to form a magnetic bead-nucleic acid complex;

[0034] Drive the external magnet to move Figure 1 downward from top to bottom on the left side of the reaction tube in the

[0035] to attract the magnetic bead-nucleic acid complex to move synchronously from top to bottom;

[0036] When moving to the amplification reaction area (i.e., the elution and amplification area 11), stay in this area for a period of time, and the magnetic bead-nucleic acid complex separates;

[0037] Drive the external magnet to move upward to attract the magnetic beads to move upward until the magnetic beads are roughly located in the middle position of the reaction tube;

[0038] During the reaction process, the optical detection probe detects the fluorescence signal at the bottom of the reaction tube to obtain the detection result. After the reaction ends, all components in the detection device are reset.

[0039] As can be seen from the above, this integrated reaction tube has a three-section structure and there is no other structure inside the tube body. Therefore, it has the advantages of simple structure and convenient production. Moreover, during the detection process, the washing movement direction of the magnetic bead-nucleic acid complex is the same as its transmission direction between adjacent sections, both in the up and down direction. This enables the external magnet that drives the movement of the magnetic beads to only move in one direction. Therefore, it also has the characteristic of a simple magnetic body movement structure for the required supporting detection equipment. Using this integrated reaction tube, the entire extraction and amplification process can be completed in one reaction tube, which can effectively avoid environmental pollution and can achieve fully automatic nucleic acid extraction, amplification, and detection. The semi-spherical structure 4 at the bottom can condense the detection light, which is beneficial to improving the detection accuracy.

[0040] In addition, in the right-angled frustum design and right-angled cone design of the reaction tube described above, on the one hand, the cracking zone 9 and the elution and amplification zone 11 respectively form a single-sided contraction structure, which is beneficial to increasing the capacity of the cracking zone 9 and saving the usage amount of the reaction solution. On the other hand, the right-angled sides 3 of the two are on the same side and flush with the side of the cylindrical section 6, so that during the entire transfer process of the magnetic beads, the suction force of the external magnet on the magnetic beads remains consistent, and magnetic bead loss can be avoided. The heating structure 8 extends to the cracking zone 9 (right-angled frustum section 5), which can heat the cracking zone 9 and helps to crack some complex samples. The cylindrical section 6 is designed as an elongated structure, and its specifications enable the magnetic bead-nucleic acid complex to have sufficient time to remove the residual lysis solution and sample on the surface, reduce the usage amount of the washing solution, and improve the stability of the entire filling liquid stratification system.

[0041] The present invention has been described in detail through specific embodiments above. These detailed descriptions are only limited to helping those skilled in the art understand the content of the present invention and should not be construed as a limitation on the protection scope of the present invention. All kinds of modifications, equivalent transformations, etc. made by those skilled in the art to the above solutions under the concept of the present invention should be included in the protection scope of the present invention.

Claims

1. An integrated reaction tube for automatic nucleic acid extraction, amplification, and detection, characterized in that, The integrated reaction tube is a test tube integrating a cracking container, a washing container, an amplification reaction container, and a detection container. Along the direction from the mouth to the bottom, the integrated reaction tube sequentially includes a right circular frustum section, a cylindrical section, and a right circular cone section. The right circular frustum section is directly connected to the cylindrical section, and the cylindrical section is directly connected to the right circular cone section. The right circular frustum section forms a cracking area, the cylindrical section forms a washing area, and the right circular cone section forms an elution and amplification area. The right sides of the right circular frustum section and the right circular cone section are on the same side and flush with the side of the cylindrical section.

2. The integrated reaction tube for automatic nucleic acid extraction, amplification, and detection according to claim 1, characterized in that, The right circular frustum section, the right circular cone section, and the cylindrical section are respectively provided with heating structures on the sides opposite to the right sides, and during the detection process, the heating structures can be fitted with the heat conduction surfaces of external heating bodies.

3. The integrated reaction tube for automatic nucleic acid extraction, amplification, and detection according to claim 2, characterized in that, The heating structure is an arc surface adapted to the reaction tube.

4. The integrated reaction tube for automatic nucleic acid extraction, amplification, and detection according to claim 1, characterized in that, The inner diameter of the cylindrical section is smaller than the average inner diameter of the right circular frustum section and larger than the average inner diameter of the right circular cone section.

5. The integrated reaction tube for automatic nucleic acid extraction, amplification, and detection according to claim 4, characterized in that, The inner diameter of the cylindrical section is 2 to 10 mm, and the ratio of the length to the inner diameter of the cylindrical section is greater than 5.

6. The integrated reaction tube for automatic nucleic acid extraction, amplification, and detection according to claim 1, characterized in that, The bottom of the integrated reaction tube includes a semi-spherical structure, and the mouth of the integrated reaction tube includes a flip cover integrally formed with the integrated reaction tube.

7. A columnar detection card, characterized in that: It includes a lysis solution, a washing solution, a reaction solution, a phase change separator, and a reaction tube; The reaction tube is the integrated reaction tube according to any one of claims 1 to 6; The lysis solution, the washing solution, and the reaction solution are respectively contained in the cracking area, the washing area, and the elution and amplification area of the integrated reaction tube. Phase change separators are respectively arranged between the lysis solution and the washing solution, and between the washing solution and the reaction solution. The mouth of the integrated reaction tube is sealed by an integrally formed flip cover. The phase change separator is in a solid state at the storage temperature and turns into a liquid state at the reaction temperature.

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