CN214218716U - Totally-enclosed integrated reaction tube capable of realizing step-by-step reaction - Google Patents
Totally-enclosed integrated reaction tube capable of realizing step-by-step reaction Download PDFInfo
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- CN214218716U CN214218716U CN202020792969.8U CN202020792969U CN214218716U CN 214218716 U CN214218716 U CN 214218716U CN 202020792969 U CN202020792969 U CN 202020792969U CN 214218716 U CN214218716 U CN 214218716U
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Abstract
The utility model discloses a totally closed integral type reaction tube that can realize substep reaction. The reaction tube body is divided into a left tube cavity and a right tube cavity by a vertical solid isolation layer, a gap is reserved between the solid isolation layer and the bottom of the tube body, the left tube cavity and the right tube cavity are communicated in a hollow mode through the bottom of the solid isolation layer, and the step-by-step reaction of a left tube cavity reaction system and the right tube cavity reaction system can be realized by combining a temperature-change phase solid filling layer arranged between the solid isolation layer and the tube wall. The utility model discloses the simple operation, it is compatible strong, use extensively, be particularly suitable for the one-tube substep reaction that needs totally closed multisystem.
Description
Technical Field
The utility model belongs to the technical field of biological experiment instruments, and relates to a totally-enclosed integrated reaction tube capable of realizing step-by-step reaction; in particular to a totally-enclosed integrated reaction tube which can effectively avoid pollution in the process of nucleic acid amplification.
Background
With the continuous development of gene amplification technology and the excavation and application of novel nucleases, the combined application of a plurality of reaction systems is more and more extensive, such as the combined reaction of PCR pre-amplification and amplification product detection, the combined reaction of nucleic acid extraction and PCR, the combined reaction of isothermal amplification technology and Argonaute protein, the combined reaction of RPA (recombinase polymerase amplification technology) and CRISPR-Cas 13 protein, and the like. However, most systems are different in reaction conditions and low in component compatibility, so that the whole-closed reaction without opening the cover cannot be performed in the same PCR tube, nucleic acid pollution is easily caused, and the operation is complicated and the time is long. Therefore, some researchers at home and abroad also propose to combine the gene chip with the microfluid technology to realize the integration of all operation processes, but because the preparation of the chip is difficult and a special reactor needs to be developed to be matched with the chip, the cost is high, and the operation difficulty is high.
Prior patent document CN101787347 discloses a reaction tube in which the progress of the reaction is controlled by temperature; this patent requires a two-step procedure, i.e., inverting the reaction tube and mixing it immediately after the first reaction time. In practical application, a fixed reaction time is usually required to be set for the first-step reaction system (exceeding the reaction time may cause experimental failure), and an operator must perform reverse mixing of the reaction tube at a fixed time point, which increases interference of human factors and increases risks caused by human errors.
Therefore, there is still a need in the art for a device that is simple, fast, and inexpensive to operate, and that is effective in avoiding contamination during nucleic acid amplification. Therefore, the utility model provides a totally closed integral type reaction tube that can realize substep reaction.
SUMMERY OF THE UTILITY MODEL
An object of the utility model is to provide a totally closed integral type reaction tube that can realize substep reaction. The utility model discloses the simple operation, it is compatible strong, use extensively, be particularly suitable for the one-tube substep reaction that needs totally closed multisystem.
The purpose of the utility model is realized through the following technical scheme:
the utility model relates to an integrated reaction tube, which comprises a tube body 2 and a tube cover 3, and further comprises an entity isolation layer 1, wherein the entity isolation layer 1 is vertically arranged in the tube body 2 and is connected with the tube wall into a whole, and divides the tube body 2 into a left semi-closed cavity and a right semi-closed cavity, namely a first cavity 5 and a second cavity 6, and a gap is reserved between the entity isolation layer 1 and the bottom of the tube body 2; the bottom of the second chamber 6 is provided with a temperature-change phase solid filling layer 4.
As an embodiment of the utility model, the space that body isolation layer 1 bottom and body 2 are located between the lateral wall of 6 sides in second cavity in body 2 is equipped with temperature change looks solid filling layer 4, seals 6 bottoms in the second cavity.
As an embodiment of the utility model, the melting point of the temperature-variable phase solid adopted by the temperature-variable phase solid filling layer 4 is 50-105 ℃, and the density range is 0.6-0.99 g/cm3And after melting, the mixture is transparent liquid.
As an embodiment of the utility model, temperature change looks solid filling layer 4 is compound paraffin filling layer, rosin filling layer, low molecular weight polyethylene filling layer, palm wax filling layer, earth wax filling layer, hot melt adhesive filling layer or polyvinyl fluoride filling layer.
As an embodiment of the utility model, the thickness of temperature change phase solid filling layer 4 is 1 ~ 3 mm.
As an embodiment of the utility model, the distance of 1 bottom of entity isolation layer and 2 bottom inner walls of body is 2 ~ 5 mm. More preferably, the distance between the bottom of the solid isolation layer 1 and the inner wall of the bottom of the tube body 2 is 3 mm.
As an embodiment of the utility model, the solid isolation layer 1 is a hexagon, the vertical height is 5-15mm, the width is 3-7mm, and the thickness is 0.2-1 mm. Preferably, the vertical height of the solid isolation layer 1 is 12mm, the upper width is 5.4mm (consistent with the inner diameter of the pipe body), the lower width is 3mm, and the thickness is 0.5 mm.
As an embodiment of the present invention, the capacity of the tube body 2 is 190 to 210 μ l. Preferably, the volume of the tube 2 is 200. mu.l.
As an embodiment of the present invention, the solid isolation layer 1 is perpendicular to the bottom of the pipe body 2.
As an embodiment of the present invention, the solid isolation layer 1 is made of, but not limited to, polypropylene, and the solid isolation layer 1 is a polypropylene isolation layer; the pipe body 2 is a polypropylene pipe body; the pipe cover 3 is a polypropylene pipe cover.
As an embodiment of the present invention, the lower portion of the tube cover 3 is provided with an outward annular protrusion. When the annular bulge enters the pipe body 2, the pipe wall extrudes the annular bulge, and the sealing performance is improved.
As an embodiment of the present invention, the pipe body 2 includes a tubular main body and a tapered bottom connected to the main body, and the lower end surface of the tapered bottom is an arc surface.
Compared with the prior art, the utility model discloses following beneficial effect has:
1) by adopting the device of the utility model, the reaction of two systems can be carried out in the same tube, the whole process is totally closed and is finished under the covering of the temperature-phase-change solid filling layer, a heating cover is not needed, and additional sealing liquid such as mineral oil is not needed to be added, thus effectively preventing the evaporation of components and the leakage of nucleic acid amplification products, avoiding aerosol pollution and effectively avoiding the pollution in the nucleic acid amplification and detection processes;
2) the operation is simple, convenient and quick: the two reaction systems are mixed in the midway without pipetting, centrifuging, shaking, reversing the reaction tube or other modes, so that the operation steps and the experiment time are greatly reduced;
3) the cost is low, and expensive additional reagents and precise instruments are not needed;
4) the device only needs one-step operation in the whole process, does not need to move liquid, centrifuge, shake and reverse the reaction tube in the reaction process, does not need to rely on other instruments or external force to realize the uniform mixing of different systems, simplifies the steps, and avoids the interference of human factors.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
fig. 1 is a schematic three-dimensional perspective structure of an embodiment of the present invention;
fig. 2 is a schematic top sectional view of a pipe body according to an embodiment of the present invention;
fig. 3 is a schematic side sectional view of an embodiment of the present invention;
FIG. 4 shows the result of the present invention applied to a portable constant temperature fluorescence detector;
wherein, 1 is the entity isolation layer, 2 is the body, 3 is the tube cap, 4 is the temperature change looks solid filling layer, 5 is first cavity, 6 is the second cavity.
Detailed Description
The present invention will be described in detail with reference to the following examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that numerous modifications and improvements can be made by those skilled in the art without departing from the inventive concepts herein. These all belong to the protection scope of the present invention.
In the present application, unless otherwise indicated, the scientific terms and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, molecular biology, enzymology, and immunology laboratory procedures used herein are all routine procedures widely used in the relevant fields. Meanwhile, in order to better understand the present disclosure, definitions and explanations of related terms are provided below.
As used herein, the term "PCR" is to be understood in a broad sense, including from all PCR (polymerase chain reaction) based gene amplification methods, including, but not limited to, reverse transcription PCR, fluorescent quantitative PCR, asymmetric PCR, nested PCR, and the like.
As used herein, the term "isothermal amplification" refers to amplification of a target gene at a constant temperature.
As used herein, the term "fully enclosed and integrated" means that the left and right lumens are respectively added with different reaction systems, and then covered with a tube cover to form a completely enclosed tube.
Example 1
A reaction tube as shown in fig. 1, fig. 2 and fig. 3, including entity isolation layer 1, body 2 and the tube cap 3 that is located the top and can open, tube cap 3 is split type structure with body 2, is made by the polypropylene material, and body 2 includes at the bottom of pipy main part and the toper that is connected with the main part, and the lower terminal surface at the bottom of the toper is the arc surface, and the lower part of tube cap 3 is equipped with annular arch outwards, and when annular arch got into in the body 2, the pipe wall played the extrusion to annular arch, improved the leakproofness. The tube body 2 is divided into a left tube cavity and a right tube cavity by the solid isolation layer 1, namely a first cavity 5 and a second cavity 6, the maximum volume is 200 mu l, and the volume of the tube body can meet the volume of most reaction systems. Entity isolation layer 1 is made by the polypropylene materials, for the hexagon, and vertical height is 12mm, and the upper portion width is 5.4mm (unanimous with the body internal diameter), and the lower part width is 3mm, and thickness is 0.5mm, and the colour is transparent, does not influence the fluorescence value reading in the reaction process. The distance between the bottom of the solid isolation layer 1 and the inner wall of the bottom of the tube body 2 is 3 mm. The space between the bottom end of the body isolation layer 1 in the pipe body 2 and the side wall of the pipe body 2, which is located on the side of the second cavity 6, is provided with a temperature change phase solid filling layer 4, the temperature change phase solid filling layer 4 is made of composite paraffin, the thickness of the composite paraffin is 2mm, the melting point of the composite paraffin is 50-105 ℃, the composite paraffin is transparent liquid after being melted, the temperature change phase solid filling layer 4 seals the bottom of the second cavity (6), and after the pipe cover 3 is covered, two semi-closed cavities with communicated tops are formed.
Example 2
A totally-enclosed integrated reaction tube capable of realizing multi-system compatibility is implemented in the fluorescence detection of SARS-CoV-2 nucleic acid isothermal amplification:
The 6 specific primer sequences are:
| F3 | TGTTCTTGCTCGCAAACA |
| B3 | GTGTTGTAAATTGCGGACAT |
| FIP | ACACATGACCATTTCACTCAATACT-AGCTTGTCACACCGTTTC |
| BIP | CATTTGTCAAGCTGTCACGGC-GCAATTTTGTTACCATCAGTAG |
| LF | CACTCATTAGCTAATCTATA |
| LB | CAATGTTAATGCACTTTT |
the isothermal amplification system is as follows:
the nucleotide DNA and the fluorescent probe sequence are as follows:
the fluorescence detection system is as follows:
and 3, setting a reaction program in a constant-temperature fluorescence detector or a fluorescence PCR instrument, wherein the first step is isothermal amplification reaction at 62 ℃ for 30 minutes, and the second step is gene editing fluorescence detection at 95 ℃ for 30 minutes. At the beginning of the second reaction, the temperature-change phase solid filler layer is melted, and the fluorescence detection system is dropped to mix with the amplification product of the first reaction.
And 4, reading the fluorescence value in real time during the second step reaction period, and judging the result as shown in figure 4, wherein 1-6 are SARS-CoV-2 nucleic acid samples with known concentration decreasing, 7 and 8 are negative controls, and the 1-5 concentration gradient detection result is positive.
The foregoing description of the specific embodiments of the invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by those skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (9)
1. A totally-enclosed integrated reaction tube capable of realizing step-by-step reaction comprises a tube body (2) and a tube cover (3), and is characterized by further comprising an entity isolation layer (1), wherein the entity isolation layer (1) is vertically arranged in the tube body (2) and is connected with the tube wall into a whole, the tube body (2) is divided into a left semi-enclosed cavity and a right semi-enclosed cavity, namely a first cavity (5) and a second cavity (6), and a gap is reserved between the entity isolation layer (1) and the bottom of the tube body (2); and a temperature-change phase solid filling layer (4) is arranged at the bottom of the second chamber (6).
2. The totally-enclosed integrated reaction tube capable of realizing step-by-step reaction according to claim 1, wherein the melting point of the temperature-change phase solid adopted by the temperature-change phase solid filling layer (4) is 50-105 ℃, and the density is 0.6-0.99 g/cm3And after melting, the mixture is transparent liquid.
3. The totally-enclosed integrated reaction tube capable of realizing step-by-step reaction according to claim 1, wherein the thickness of the temperature-change phase solid filling layer (4) is 1-3 mm.
4. The totally-enclosed integrated reaction tube capable of realizing step-by-step reaction according to claim 1, wherein the distance between the bottom of the solid isolation layer (1) and the inner wall of the bottom of the tube body (2) is 2-5 mm.
5. The totally-enclosed integrated reaction tube capable of realizing step-by-step reaction according to claim 1, wherein the solid isolation layer (1) is a hexagon, the vertical height is 5-15mm, the width is 3-7mm, and the thickness is 0.2-1 mm.
6. The totally-enclosed integrated reaction tube capable of realizing step-by-step reaction according to claim 1, wherein the volume of the tube body (2) is 190-210 μ l.
7. The totally-enclosed integrated reaction tube capable of realizing step-by-step reaction according to claim 1, wherein the solid isolation layer (1) is perpendicular to the bottom of the tube body (2).
8. The totally-enclosed integrated reaction tube capable of realizing step-by-step reaction according to claim 1, wherein the lower part of the tube cover (3) is provided with an outward annular bulge.
9. The totally-enclosed integrated reaction tube capable of realizing step-by-step reaction according to claim 1, wherein the tube body (2) comprises a tubular main body and a conical bottom connected with the main body, and the lower end surface of the conical bottom is a circular arc surface.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202020792969.8U CN214218716U (en) | 2020-05-14 | 2020-05-14 | Totally-enclosed integrated reaction tube capable of realizing step-by-step reaction |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202020792969.8U CN214218716U (en) | 2020-05-14 | 2020-05-14 | Totally-enclosed integrated reaction tube capable of realizing step-by-step reaction |
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| CN214218716U true CN214218716U (en) | 2021-09-17 |
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| CN202020792969.8U Expired - Fee Related CN214218716U (en) | 2020-05-14 | 2020-05-14 | Totally-enclosed integrated reaction tube capable of realizing step-by-step reaction |
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2020
- 2020-05-14 CN CN202020792969.8U patent/CN214218716U/en not_active Expired - Fee Related
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Granted publication date: 20210917 |