CN216550469U

CN216550469U - Test tube for pre-sealing and storing lysis solution for quickly extracting nucleic acid

Info

Publication number: CN216550469U
Application number: CN202120988379.7U
Authority: CN
Inventors: 王德明; 周杰锋
Original assignee: Ningbo Kangcheng Deno Biomedical Co ltd
Current assignee: Ningbo Kangcheng Deno Biomedical Co ltd
Priority date: 2021-05-10
Filing date: 2021-05-10
Publication date: 2022-05-17
Anticipated expiration: 2031-05-10

Abstract

The application relates to the technical field of nucleic acid extraction, in particular to a test tube for pre-sealing and storing a lysis solution for quickly extracting nucleic acid. A test tube for pre-sealing a lysis solution for quickly extracting nucleic acid comprises a tube body, wherein the bottom inside the tube body is pre-sealed with nano magnetic beads and nucleic acid extraction proteolytic enzyme (21), an isolation layer (22) is arranged above the nano magnetic beads and the nucleic acid extraction proteolytic enzyme (21), and other lysis solution components (23) except the nucleic acid extraction proteolytic enzyme are sealed above the isolation layer (22); the isolation layer (22) is made of organic solid materials with the melting point of 40-65 ℃. This application separates lysate and nanometer magnetic bead and nucleic acid extraction albumen lytic enzyme in the lysate test tube with solid-state separate layer, then through heating melt separate layer, utilizes liquid density to make lysate and nanometer magnetic bead and nucleic acid extraction albumen lytic enzyme contact.

Description

Test tube for pre-sealing and storing lysis solution for quickly extracting nucleic acid

Technical Field

The application relates to the technical field of nucleic acid extraction, in particular to a test tube for pre-sealing and storing a lysis solution for quickly extracting nucleic acid.

Background

The real-time fluorescent quantitative PCR technology is widely applied to a plurality of fields such as genetic disease molecular diagnosis, clinical examination, animal and plant import and export quarantine, food safety monitoring, soil microorganism detection, paternity test and the like. Because samples such as blood, food, soil and the like contain a large amount of inhibiting factors such as hemoglobin, methemoglobin, lactoferrin, humic acid and the like, the Taq DNA polymerase has obvious inhibiting effect on the conventional Taq DNA polymerase. Therefore, nucleic acids must be isolated from these test samples and then used for PCR amplification. Nucleic acid extraction is the first step of nucleic acid detection, and is also one of the key methods in molecular biology. Provides a basis for downstream nucleic acid detection, and the quality and integrity of extraction directly influence clinical research or diagnosis.

The general nucleic acid extraction process comprises three steps: lysis, rinsing and elution processes. The traditional extraction reagent has relatively low extraction rate and complex steps, thus bringing certain influence on extraction work, and particularly being difficult to obtain qualified nucleic acid when the sample amount is rare. In addition, the traditional method has the disadvantages of multiple operation steps, high cost, large sample requirement, easy cross contamination and inconvenience for customers to quickly extract and purify nucleic acid samples.

In addition, in the current real-time fluorescence PCR technology with the widest application of nucleic acid detection, most reagents are not ready-to-use, so that the problems of complicated operation, high labor and cost and the like of PCR detection generally exist, complicated configuration work is required before use, and errors and even failures of tests are easily caused. Experimental errors also arise from different personnel operations; the rapid extraction of nucleic acid by one-step method cannot be realized; PCR detection reagents need to be manually configured on site, multiple steps need to be manually marked, detection instruments at all stages are independent, and full-automatic treatment of a large number of test samples is not facilitated. Particularly, in field work (such as workers in livestock breeding industry and pet disease diagnosis and examination), the sample is not easy to preserve, and the nucleic acid substances in the sample can be decomposed over time, so that the subsequent detection is not facilitated.

In general, a lysis solution for nucleic acid extraction needs to be added with a nucleic acid extraction proteolytic enzyme, such as proteinase K, etc., and the nucleic acid extraction proteolytic enzyme is a bioactive component and is not easy to store for a long time after being mixed with other lysis solutions. The nucleic acid extracting proteolytic enzyme of the present application may be added to the lysate at the time of use, but the most convenient method is to seal the lysate tube, which is more convenient to use.

Disclosure of Invention

In order to solve the above technical problems, an object of the present application is to provide a pre-sealed storage tube for a rapid nucleic acid extraction lysate, in which a solid separation layer is used to separate the lysate from the nano magnetic beads and the nucleic acid extraction proteolytic enzyme in the lysate tube, and then the lysate is contacted with the nano magnetic beads and the nucleic acid extraction proteolytic enzyme by heating and melting the separation layer and using the liquid density.

In order to achieve the purpose, the bottom inside a lysate test tube is sealed with nano magnetic beads and nucleic acid extraction proteolytic enzyme in advance, an isolation layer is arranged above the nano magnetic beads and the nucleic acid extraction proteolytic enzyme, and other lysate components except the nucleic acid extraction proteolytic enzyme are sealed above the isolation layer; the isolating layer is made of organic solid materials with the melting point of 40-65 ℃.

Preferably, the nucleic acid extraction proteolytic enzyme is proteinase K or pepsin; the isolating layer adopts paraffin; proteinase K is most preferred in this application, and paraffin is also most preferred.

Preferably, the wall thickness of the bottom of the lysate test tube is increased to form a hemispherical cavity; the nanometer magnetic beads and the nucleic acid extraction protein lytic enzyme are stored in the hemispherical cavity.

This application is owing to adopted foretell technical scheme, and this application separates lysate and nanometer magnetic bead and nucleic acid extraction proteolytic enzyme in with solid-state separate layer in the lysate test tube, then through heating melt separate layer, utilizes liquid density to make lysate and nanometer magnetic bead and nucleic acid extraction proteolytic enzyme contact. The method really realizes the rapid extraction of nucleic acid, is simple, rapid and efficient, and is particularly suitable for services such as field sampling, pet disease detection and the like.

Drawings

Fig. 1 is a schematic structural diagram of the quadruple pipe device of the present application.

FIG. 2 is a schematic view of the structure of the stirring rod of the present application.

Fig. 3 is a schematic top view of the quadruple-tube device according to the present application.

Fig. 4 is a cross-sectional view of fig. a-a.

FIG. 5 is a contrast graph of the detection rate of the present application and the conventional magnetic bead extraction.

Detailed Description

The following detailed description of embodiments of the present application refers to the accompanying drawings.

As shown in FIG. 1 and FIG. 3, the four-way pipe device for rapid extraction of nucleic acid is integrally molded by one of PE, PP, PA, PC, PPS, PET, PVC and PMMA. The device includes first panel 1, lysate test tube 2, first rinsing liquid test tube 3, second rinsing liquid test tube 4, second panel 5 and eluant test tube 6, lysate test tube 2, first rinsing liquid test tube 3 and second rinsing liquid test tube 4 set up on first panel 1, second panel 5 sets up the end plate at first panel 1, is provided with connecting portion 7 that enables first panel 1 and 5 rupture of second panel between first panel 1 and second panel 5, as shown in fig. 1, connecting portion 7 sets up the scale or breach for connecting the connecting strip of the reduction between first panel 1 and the second panel 5. The eluent tube 6 is disposed on the second panel 5; the lysis solution, the first rinsing solution, the second rinsing solution and the eluent for extracting nucleic acid are respectively sealed in advance in the lysis solution test tube 2, the first rinsing solution test tube 3, the second rinsing solution test tube 4 and the eluent test tube 6.

As shown in fig. 4, the bottom inside the lysis solution test tube 2 is pre-sealed with nano magnetic beads and nucleic acid extraction proteolytic enzyme 21, an isolation layer 22 is arranged above the nano magnetic beads and nucleic acid extraction proteolytic enzyme 21, and other lysis solution components 23 except for nucleic acid extraction proteolytic enzyme are sealed above the isolation layer 22; the isolating layer 22 is made of organic solid materials with the melting point of 40-65 ℃. The nucleic acid extraction protein lytic enzyme is selected from proteinase K or pepsin; the isolating layer 22 adopts paraffin; the second rinsing liquid does not contain ethanol. The wall thickness of the bottom of the lysate test tube 2 is increased to form a hemispherical cavity; the nanometer magnetic beads and the nucleic acid extraction protein lytic enzyme are stored in the hemispherical cavity. The bottom of the eluent test tube 6 is a centrifugal tube 61, and a support sleeve 62 is arranged outside the centrifugal tube 61. So that the folded test tube can be conveniently prevented.

As shown in fig. 1, the device further comprises a first cover plate 8, the first cover plate 8 is provided with three end covers 9 which are matched with the lysate test tube 2, the first rinsing liquid test tube 3 and the second rinsing liquid test tube 4, the first cover plate 8 is connected with one side of the first panel 1 through a first turnover connecting part 10, the first cover plate 8 can be turned over to the top of the first panel 1 through the first turnover connecting part 10, and the three end covers 9 are respectively covered on the lysate test tube 2, the first rinsing liquid test tube 3 and the second rinsing liquid test tube 4. A first panel bulge 11 is arranged at a first side position of the first panel 1 facing the first cover plate 8, the first turnover connecting part 10 is connected with the first panel bulge 11, and a first bayonet 12 is arranged on the first panel bulge 11; and a first buckle 13 is arranged at the position of the first cover plate 8, which is folded and corresponds to the first bayonet 12.

As shown in fig. 1, the device further includes a second cover plate 14, the second cover plate 14 is provided with an end cap 9 mutually matched with the eluent test tube 6, and the second cover plate 14 is connected with one side of the second panel 5 through a second folding connecting portion 15, and the second cover plate 14 can be folded to the upper side of the second panel 5 through the second folding connecting portion 15, so that the end caps 9 are respectively covered on the eluent test tube 6. A second panel bulge 16 is arranged at a first side position of the second panel 5 facing the second cover plate 14, the second turnover connecting part 15 is connected with the second panel bulge 16, and a second bayonet 17 is arranged on the second panel bulge 16; and a second buckle 18 is arranged at the position of the second cover plate 14, which is folded and corresponds to the second bayonet 17.

As shown in fig. 1, a third buckle 19 is arranged at a second side edge of the first cover plate 8, which is turned over and corresponds to the first panel 1 and faces away from the first cover plate 8, and the third buckle 19 is turned over and buckled at the second side edge. A fourth buckle 10 is arranged at a second side edge of the second cover plate 14, which is turned over and corresponds to the second panel 5 and is back to the second cover plate 14, and a fourth buckle 20 is turned over and buckled at the second side edge.

Referring to fig. 1 and 2, a nucleic acid rapid extraction kit of the present application includes the quadruple tube device, a stirring rod with a magnetic head 30 at an end thereof, and magnetic beads; the magnetic beads are arranged separately or added in advance into the lysis solution test tube 2. The stirring rod comprises a handle 32, an outer sleeve 31 and the magnetic head 30, wherein the magnetic head 30 is arranged at the bottom of the outer sleeve 31 in a closed mode, and the upper portion of the outer sleeve 31 is fixedly connected with the handle 32.

The method for rapidly extracting nucleic acid adopts the kit and comprises the following steps:

1) placing the lysis solution test tube 2 into a water bath or a metal bath, raising the temperature until paraffin is dissolved, and fully mixing the nucleic acid extraction protein lytic enzyme and lysis solution magnetic beads;

2) adding the biological sample into a lysis solution test tube containing magnetic beads, nucleic acid extraction protein lytic enzyme and lysis solution for lysis and releasing nucleic acid, and adsorbing the released nucleic acid by using nano magnetic beads;

3) stretching a stirring rod into a lysate test tube to draw up and down or stir, and adsorbing magnetic beads to the magnetic end part of the outer sleeve of the stirring rod;

4) the stirring rod adsorbed with the magnetic beads sequentially enters a first rinsing liquid test tube and a second rinsing liquid test tube to be pumped and rinsed up and down without being dried;

5) the puddler that the rinsing was accomplished directly gets into the eluant test tube and elutes, and the back is accomplished in the elution, breaks the second panel from first panel, and lysate test tube, first rinsing liquid test tube and second rinsing liquid test tube abandonment on first panel and the first panel are preserved or direct sample with the eluant test tube low temperature and are detected.

By collecting nucleic acid substances of pig blood, river crab tissues and cat ascites and comparing the method with the traditional magnetic bead method, the detection rate is obviously improved, and the result is shown in figure 5.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present disclosure, including any person skilled in the art, having the benefit of the present disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A test tube for pre-sealing a lysis solution for quickly extracting nucleic acid comprises a tube body and is characterized in that a nano magnetic bead and a nucleic acid extraction proteolytic enzyme (21) are pre-sealed at the bottom inside the tube body, an isolation layer (22) is arranged above the nano magnetic bead and the nucleic acid extraction proteolytic enzyme (21), and other lysis solution components (23) except the nucleic acid extraction proteolytic enzyme are sealed above the isolation layer (22); the isolation layer (22) is made of organic solid materials with the melting point of 40-65 ℃.

2. The test tube for pre-sealing and storing the nucleic acid rapid extraction lysate according to claim 1, wherein the nucleic acid extraction proteolytic enzyme is proteinase K or pepsin.

3. The test tube for pre-sealing and storing the nucleic acid rapid extraction lysate according to claim 1, wherein the isolation layer (22) is paraffin.

4. The test tube for pre-sealing and storing the lysis solution for the rapid extraction of the nucleic acid according to claim 1, wherein the wall thickness of the bottom of the tube body is increased to form a hemispherical chamber; the nanometer magnetic beads and the nucleic acid extraction protein lytic enzyme (21) are stored in the hemispherical cavity.