CN216688113U

CN216688113U - Constant temperature nucleic acid amplification reaction tube

Info

Publication number: CN216688113U
Application number: CN202120494028.0U
Authority: CN
Inventors: 黄婉秋; 黄健
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-03-03
Filing date: 2021-03-03
Publication date: 2022-06-07
Anticipated expiration: 2031-03-03

Abstract

The utility model discloses a constant-temperature nucleic acid amplification reaction tube which comprises a tube body, a tube cover and a puncture part. The constant-temperature nucleic acid amplification reaction tube can effectively avoid aerosol pollution, obviously improves the accuracy of a detection result, and has a good application prospect.

Description

Constant temperature nucleic acid amplification reaction tube

Technical Field

The utility model relates to the technical field of medical detection tools, in particular to a constant-temperature nucleic acid amplification reaction tube.

Background

Recombinase Polymerase Amplification (RPA) is based on the principle of Recombinase Polymerase-mediated Amplification, simulates the enzymatic reaction process of in vivo DNA replication, relies on specific enzyme and protein combinations (Recombinase, single-strand binding protein and DNA Polymerase) to perform specific Amplification on a DNA template, and is a constant-temperature nucleic acid Amplification technology, can realize rapid specific amplification (25-42 ℃, 5-30min) under the condition of approaching body temperature, reduces the dependence on high-precision expensive instruments, stable power supply facilities and high-grade laboratories as an isothermal technology, can be completed by only needing a constant temperature device, and whether the amplification product exists or not can be judged through the fluorescence analysis device, so that the method has the characteristic of simpler facility and operation requirements, and is widely applied to multiple fields of life science research, medical detection, agriculture, food safety, transgenic detection and the like.

In order to further improve The detection sensitivity of RPA, researchers have proposed a nested RPA detection method (nestRPA) (Wanqiu Huang, Dachuan Lin, Cuini Wang, Chaohui Bao, Zhaoqi Zhang, Xinchun Chen, Zheng Zhang, Jian Huang #, The determination of release from isolation of COVID-19 substrates require ultra-high sensitivity nucleic acid technology. J Infect.2020Jul 2; S0163-4453(20) 56-4.doi:10.1016/j. jinf. 2020.06.075), which basically: a first segment of the target gene is amplified using the outer primers, and then a second segment of the target gene, which is completely within the first amplified segment, is amplified using the inner primers. In order to eliminate the effect of the enzyme's fluorescent signal, no fluorescent probe was included in the first RPA reaction system. In the second RPA reaction, a fluorescent probe was added. Experiments prove that the nestRPA has ultrahigh sensitivity, and provides powerful technical support for early discovery of various viruses such as new coronavirus, influenza virus, classical swine fever virus and the like. However, the nestRPA needs to be subjected to two times of RPA reactions, after the first time of RPA reaction is finished, the cover of the reaction tube needs to be opened, the inner primer and the probe needed by the second time of RPA reaction are added, and then the cover of the reaction tube is covered to carry out the second time of RPA reaction until the detection result of the reaction is finished. Among them, in the process of opening the cap of the reaction tube after the first RPA reaction is completed, nucleic acid aerosol contamination is easily generated, resulting in false positive results. Therefore, there is an urgent need to develop a reaction tube which can complete the nestRPA in a "one-tube method" and perform the second RPA reaction without opening the cap of the reaction tube after the first RPA reaction.

SUMMERY OF THE UTILITY MODEL

The utility model aims to solve the technical problem that the prior reaction tube for nested RPA detection is easy to generate nucleic acid aerosol pollution, and provides a constant-temperature nucleic acid amplification reaction tube which can effectively avoid uncapping operation in the process of nested RPA detection, prevent pollution and enable the detection result to be more accurate.

In order to solve the technical problems, the utility model is realized by the following technical scheme:

in one aspect of the present invention, there is provided a isothermal nucleic acid amplification reaction tube including a tube body, a tube cap, and a piercing part disposed inside an inlet port of the tube body.

Preferably, the puncture part is in the shape of a hollow cone, and a plurality of holes are formed in the side surface of the cone, so that the liquid can sink to the bottom of the tube after centrifugation.

The opening of the tube cap can be sealed by a sealing film, and the sealing film comprises a heat-resistant film type sealing film.

The top end of the tube cover can be provided with a sample adding port.

On the premise that the bottom of the tube cover is sealed in advance and can puncture the sealing film, reaction liquid can be added into the tube cover through the sample adding port at the top end of the tube cover.

In another aspect of the present invention, there is provided a isothermal nucleic acid amplification reaction tube according to another embodiment, comprising a tube body, a tube cap, and a piercing member disposed at a tip of the tube cap.

Preferably, the piercing member is a nail-shaped member penetrating the tip of the cap, and the piercing member is pushed outside the tip of the cap.

Liquid such as a PCR reaction system and the like can be added into the tube cap, and the opening of the tube cap can be sealed by a sealing film.

According to the constant-temperature nucleic acid amplification reaction tube, through a unique structural design, the nested RPA detection can be carried out for the second time without opening the cover of the reaction tube after the first time of RPA reaction, so that aerosol pollution is effectively avoided, and the accuracy of a detection result is obviously improved.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a schematic perspective view of a isothermal nucleic acid amplification reaction tube according to the present invention;

FIG. 2 is an exploded view of the isothermal nucleic acid amplification reaction tube according to the present invention;

FIG. 3 is a schematic external view of the isothermal nucleic acid amplification reaction tube of the present invention;

FIG. 4 is a schematic perspective view of another embodiment of the isothermal nucleic acid amplification reaction tube of the present invention;

wherein, in fig. 1-4:

1-tube body, 2-tube cover, 3-puncture component and 4-puncture component.

Detailed Description

As shown in FIG. 1-2, the isothermal nucleic acid amplification reaction tube of the present invention comprises a tube body 1, a cap 2, and a piercing member 3, wherein the piercing member 3 is disposed inside an inlet of the tube body 1. In one embodiment of the utility model, the piercing member 3 is in the shape of a hollow cone provided with a plurality of holes 4 on its conical side (see fig. 3). When the nested RPA detection is carried out by using the reaction tube, a reaction system of the first RPA reaction is added in the tube cap 2, the opening of the tube cap 2 is sealed by a sealing film (Parafilm), and then the tube cap is placed in a constant temperature amplification instrument for carrying out the first RPA reaction at 39 ℃. When the first RPA reaction is finished, components except for a reaction template required by the second RPA reaction are added in advance in the tube body 1, the tube cover 2 sealed with a sealing film is covered in the tube body 1, the sealing film at the opening of the tube cover 2 can be punctured by the puncture part 3 arranged at the inner side of the inlet of the tube body 1 at the moment, so that the first RPA reaction product in the tube cover 2 completely flows into the tube body 1 through the hole 4 on the conical side surface of the puncture part 3 to be used as a template of the second RPA reaction, then, the mixture and the centrifugation are carried out, the second RPA reaction is carried out in a fluorescence-isothermal amplification instrument at 39 ℃, and finally, the fluorescence result is detected. By adopting the constant-temperature nucleic acid amplification reaction tube, the nest type RPA detection can be carried out for the second time without opening the cover of the reaction tube after the first time of RPA reaction is finished, so that aerosol pollution caused by uncovering operation is effectively avoided, and the accuracy of the detection result is obviously improved.

In another embodiment of the present invention, the top end of the tube cover 2 is opened with a sample addition port. When the reaction tube is used for nest type RPA detection, the opening at the lower part of the tube cap 2 is sealed by a sealing film, then a reaction system for the first RPA reaction is added from a sample adding port at the top end of the tube cap 2, the sample adding port is sealed, and then the reaction system is placed in a constant temperature amplification instrument for the first RPA reaction at 39 ℃. When the first RPA reaction is finished, components except for a reaction template required by the second RPA reaction are added in advance in the tube body 1, the tube cover 2 sealed with a sealing film is covered in the tube body 1, the sealing film at the opening of the tube cover 2 can be punctured by the puncture part 3 arranged at the inner side of the inlet of the tube body 1 at the moment, so that the first RPA reaction product in the tube cover 2 completely flows into the tube body 1 through the hole 4 on the conical side surface of the puncture part 3 to be used as a template of the second RPA reaction, then, mixing and centrifuging are carried out, the second RPA reaction is carried out in a fluorescence-constant temperature amplification instrument at 39 ℃, and finally, the fluorescence result is detected. By adopting the constant-temperature nucleic acid amplification reaction tube, the nest type RPA detection can be carried out for the second time without opening the cover of the reaction tube after the first time of RPA reaction is finished, so that aerosol pollution caused by uncovering operation is effectively avoided, and the accuracy of the detection result is obviously improved.

As shown in FIG. 4, in another embodiment of the present invention, a isothermal nucleic acid amplification reaction tube comprises a tube body 1, a cap 2, and a piercing member 3, wherein the piercing member 3 is provided at the tip of the cap 2. In one embodiment, the piercing member 3 is a nail-shaped member penetrating the top end of the cap 2, and the piercing member 3 is pushed outside the top end of the cap 2. When the reaction tube is used for nest type RPA detection, a reaction system of the first RPA reaction is added in the tube cover 2, the opening of the tube cover 2 is sealed by a sealing film (parafilm), and then the tube cover is placed in a constant temperature amplification instrument to carry out the first RPA reaction at the temperature of 39 ℃. When the first RPA reaction is finished, components except for a reaction template required by the second RPA reaction are added in advance in the tube body 1, the tube cover 2 sealed with a sealing film is covered in the tube body 1, then the tail end of the puncture component 3 on the outer side of the top end of the tube cover 2 is pushed, the sharp head of the puncture component 3 moves forward, the sealing film at the opening of the tube cover 2 is punctured, the first RPA reaction product in the tube cover 2 completely flows into the tube body 1 to be used as a template of the second RPA reaction, then mixing and centrifuging are carried out, the second RPA reaction is carried out in a fluorescence-isothermal amplification instrument at 39 ℃, and finally the fluorescence result is detected. The constant-temperature nucleic acid amplification reaction tube of the embodiment also enables the nested RPA detection to be carried out for the second RPA reaction without opening the cover of the reaction tube after the first RPA reaction is finished, thereby effectively avoiding aerosol pollution caused by uncovering operation and obviously improving the accuracy of detection results.

The above-described embodiments are specific and detailed, but should not be construed as limiting the scope of the utility model. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The constant-temperature nucleic acid amplification reaction tube is characterized by comprising a tube body (1), a tube cover (2) and a puncture part (3), wherein the puncture part (3) is arranged on the inner side of an inlet of the tube body (1), the puncture part (3) is in a hollow conical shape, and a plurality of holes (4) are formed in the conical side surface of the puncture part.

2. A constant temperature nucleic acid amplification reaction tube is characterized by comprising a tube body (1), a tube cover (2) and a puncturing part (3), wherein the puncturing part (3) is arranged at the top end of the tube cover (2), the puncturing part (3) is a nail-shaped part penetrating through the top end of the tube cover (2), and the puncturing part (3) can be pushed outside the top end of the tube cover (2).

3. The isothermal nucleic acid amplification reaction tube according to claim 1 or 2, wherein the opening of the tube cap (2) is sealable with a sealing film comprising a heat-resistant film type sealing film.

4. The isothermal nucleic acid amplification reaction tube according to claim 1 or 2, wherein a top end of the tube cover (2) is provided with a sample addition port.

5. The isothermal nucleic acid amplification reaction tube according to claim 4, wherein the tube cap (2) is provided with a pierceable sealing film at the bottom thereof, and a reaction liquid is added to the interior of the tube cap (2).