PRIORITY CLAIM TO RELATED APPLICATIONS

This application is a U.S. national stage filing under 35 U.S.C. § 371 from International Application No. PCT/CN2019/074312, filed on 1 Feb. 2019, and published as WO2020/001030 on 2 Jan. 2020, which claims the benefit under 35 U.S.C. 119 to Chinese Application No. 201810669579.9, filed on 26 Jun. 2018, the benefit of priority of each of which is claimed herein, and which applications and publication are hereby incorporated herein by reference in their entirety.

TECHNICAL FIELD

The invention relates to a nucleic acid detection method and a reagent tube, in particular to a nucleic acid integrated detection method and a detection reagent tube.

BACKGROUND ART

Laboratory nucleic acid detection has a short cycle and its sensitivity and specificity is comparable with that of a culture method; at present, a nucleic acid detection reagent tube is usually used in the nucleic acid detection for a sample. In existing nucleic acid detection tubes, a lysis solution, a cleaning solution and a reaction solution are usually separated by solid separation layers which are then heated and melt so that magnetic beads carrying nucleic acid can pass through the separation layers for corresponding operations. However, the nucleic acid is sensitive to changes in temperature, thus an operator needs to strictly control a heating area to reduce influence of the changes in temperature on nucleic acid, which will increase operation difficulty. Meanwhile, the existing detection tubes are easily interfered by many factors (for example, the interference of temperature on nucleic acid), resulting in a large error in an existing detection result. Therefore, it exists problems of a large detection error and a large operational difficulty for existing technologies.

SUMMARY

An object of the present invention is to provide a nucleic acid integrated detection method and a detection reagent tube. The invention presents advantages of reduced detection errors and operational difficulty.

According to a technical scheme of the invention, a nucleic acid integrated detection method is provided, including: separating a lysis solution, a cleaning solution and a reaction solution in a detection reagent tube by providing a plurality of separation plugs arranged one above one another and disposing a hydrophobic layer in a liquid or a solid phase at each separation plug; adding a sample into the lysis solution for mixing and lysis; extracting a nucleic acid in the sample using magnetic nanobeads; and then driving, by an external magnet, the magnetic nanobeads carrying the nucleic acid to sequentially pass through each hydrophobic layer along a magnetic bead channel in an inner wall of the detection reagent tube and into the cleaning solution and the reaction solution to realize a cleaning and amplification for the nucleic acid, wherein a biological agent required in the reaction solution is stored in a separation plug above the reaction solution; and finally, detecting the nucleic acid of the sample by an external device using an optical detection method, thus realizing a plurality of steps of nucleic acid extraction, cleaning and amplification reaction in the same detection reagent tube.

In the aforementioned nucleic acid integrated detection method, one or more branch tubes are provided at a lower part of the detection tube, the amplification reaction is performed in each branch tube, and one or more clusters of magnetic nanobeads carrying the nucleic acid are driven into the one or more branch tubes by the external magnet to react independently, so that nucleic acid from one sample can be simultaneously detected in one or more different reaction systems.

In the aforementioned nucleic acid integrated detection method, a reagent storage chamber with a downward opening is disposed in the separation plug above the reaction solution, a biochemical reagent is disposed in the reagent storage chamber, the biochemical reagent is stored on a carrier, and is sealed and protected by a hydrophobic sealing layer in the reagent storage chamber.

In the aforementioned nucleic acid integrated detection method, the biochemical reagent is stored on a magnetic carrier and sealed and protected by the hydrophobic sealing layer in the reagent storage chamber; when the reaction is needed, the biological reagent is driven by the external magnet to pass through the hydrophobic sealing layer and into the reaction solution, thus realizing the transferring of the biochemical reagent from the reagent storage chamber to the reaction solution.

In the aforementioned nucleic acid integrated detection method, the biochemical reagent is stored on the carrier and sealed and protected by the hydrophobic sealing layer composed of a hot melt substance; when the reaction is needed, the separation plug is heated in a temperature control mode, in which the hot melt substance is heated and melted so that the biochemical reagent in the storage chamber can move out, thus realizing the transferring of the biochemical reagent from the reagent storage chamber to the reaction solution.

A nucleic acid integrated detection reagent tube includes a main tube, and one or more branch tubes are provided at a lower end of the main tube, wherein a lysing zone, a first separation plug, a cleaning zone and a second separation plug are sequentially disposed from top to bottom in the main tube, a reaction zone is provided in each branch tube and the second separation plug is positioned at a connection between the one or more branch tubes and the main tube; a reagent storage chamber with a downward opening is disposed in the second separation plug, a carrier for storing reagents is disposed in the reagent storage chamber and a hydrophobic sealing layer is also provided in the reagent storage chamber; hydrophobic layers in a liquid or a solid phase are disposed at both the first separation plug and the second separation plug; and a magnetic bead channel penetrating to the one or more branch tubes is also defined in an inner wall of the main tube.

In the aforementioned nucleic acid integrated detection reagent tube, a magnetizable mixing device is provided in the reaction zone and/or the cleaning zone.

In the aforementioned nucleic acid integrated detection reagent tube, the first separation plug comprises a plug wherein a tapered surface is provided on an upper end of the plug and a bump is provided above the tapered surface.

In the aforementioned nucleic acid integrated detection reagent tube, a plurality of arc-shaped convex surfaces are provided on a side wall of the plug and a channel is provided between adjacent arc-shaped convex surfaces.

In the aforementioned nucleic acid integrated detection reagent tube, the second separation plug includes a partition plate, wherein a plurality of plug bodies corresponding to positions of the one or more branch tubes are arranged below the partition plate, a protrusion is arranged on an upper end of the partition plate; and a reagent storage chamber is disposed in each plug body.

Compared with the prior art, according to the present invention, the lysis solution, the cleaning solution and the reaction solution in the detection reagent tube are separated by mutually cooperatively arranging a plurality of separation plugs and hydrophobic layers in a liquid or a solid phase, and the magnetic nanobeads are driven by the external magnet to move along the magnetic bead channel without any heating required for the detection reagent tube during the whole movement process of the magnetic nanobeads, thus not only simplifying the operation and reducing the operational difficulty, but also reducing the interference of temperature on nucleic acid, effectively improving the detection accuracy and reducing the errors; meanwhile, all reactions are concentrated in one detection reagent tube, which can effectively avoid pollution and cross contamination and improve detection accuracy. According to the invention, the lysis solution and the cleaning solution are placed in the main tube and the reaction solution in the branch tubes so that nucleic acid can enter into different branch tubes respectively after being cleaned, enabling nucleic acid entering into different branch tubes to be with the same state, which results in reduced interference and detection errors. According to the invention, the reagent storage chamber is arranged in the separation plug (i.e. the second separation plug) above the reaction solution, and a dry environment for storing the biochemical reagent is provided through the mutual cooperation of the hydrophobic layer in liquid or solid phase with the hydrophobic sealing layer in the reagent storage chamber, so that the reduced stability of biochemical reagent after being subjected to moisture can be effectively prevented, a better storage can be presented due to a packaging effect of the hydrophobic layer, and a better redissolution effect also can be obtained by using the magnetic or non-magnetic carrier as a biochemical reagent attachment, thereby effectively improving detection accuracy and reducing errors. In summary, the invention presents advantages of reduced detection errors and operational difficulty.

In addition, according to the present invention, the reagent storage chamber is arranged in the second separation plug and the hydrophobic sealing layer composed of a hot melt substance is arranged in the reagent storage chamber, so that the transfer of the biochemical reagent can be performed in a temperature control mode, and an elution and redissolution of nucleic acid can be made in different steps so as to facilitate a more thorough elution for the nucleic acid, thus effectively improving the utilization of samples and the effect of the nucleic acid amplification reaction;

A narrow channel for storing hydrophobic substance is formed between the first separation plug and the tube wall of the reagent tube, so that both a better fixing of the first separation plug and a good separation effect can be realized.

