阅读说明：本技术 一种一体化核酸检测器件及应用 (Integrated nucleic acid detection device and application ) 是由 陈柱 何农跃 李松 邓燕 周枫林 于 2019-08-22 设计创作，主要内容包括：本发明公开了一种一体化核酸检测器件及应用,包括壳体、试剂存储单元、移液单元、导轨、旋转盘和设在壳体上的探测孔,所述导轨沿壳体轴向贯穿,所述试剂存储单元和旋转盘位于导轨的下方,所述移液单元通过沿导轨运动在试剂存储单元和旋转盘之间转移液体。本发明的装置通过移液单元沿导轨运动在试剂存储单元完成一系列反应,再通过移液单元沿导轨运动与旋转盘转动相互配合,将反应后溶液分装至旋转盘内部的孔道,所述溶液和旋转盘内预装的试剂反应,完成PCR扩增及检测反应,再配合旋转盘转动使待测溶液对准探测孔,对DNA或RNA扩增产物进行检测,通过简单的操作可以在装置内完成核酸扩增中变性、退火、延伸过程,大大提高了检测速度。(The invention discloses an integrated nucleic acid detection device and application thereof, and the integrated nucleic acid detection device comprises a shell, a reagent storage unit, a pipetting unit, a guide rail, a rotating disk and a detection hole arranged on the shell, wherein the guide rail axially penetrates through the shell, the reagent storage unit and the rotating disk are positioned below the guide rail, and the pipetting unit transfers liquid between the reagent storage unit and the rotating disk by moving along the guide rail. The device completes a series of reactions in the reagent storage unit by the movement of the liquid transfer unit along the guide rail, then the movement of the liquid transfer unit along the guide rail is matched with the rotation of the rotating disc, the solution after the reactions is subpackaged to the pore channel in the rotating disc, the solution reacts with the reagent preinstalled in the rotating disc to complete PCR amplification and detection reactions, and then the rotation of the rotating disc is matched to enable the solution to be detected to be aligned to the detection hole to detect the DNA or RNA amplification product.)

1. An integrated nucleic acid detecting device characterized in that: the device comprises a housing (104), a reagent storage unit, a pipetting unit, a guide rail (103), a rotating disc (108) and a detection hole (106) arranged on the housing (104), wherein the guide rail (103) penetrates through the housing (104) in the axial direction, the reagent storage unit and the rotating disc (108) are positioned below the guide rail (103), and the pipetting unit transfers liquid between the reagent storage unit and the rotating disc (108) through moving along the guide rail (103).

2. The integrated nucleic acid detecting device according to claim 1, characterized in that: the device also comprises a liquid transfer pump (102) and an air guide tube (101), wherein the liquid transfer unit is connected with the liquid transfer pump (102) through the air guide tube (101).

3. The integrated nucleic acid detecting device according to claim 1 or 2, characterized in that: the device also comprises a motor for driving the guide rail (103) to rotate, the guide rail (103) is a lead screw, the pipetting unit is sleeved on the guide rail (103), and the guide rail (103) is driven to rotate by the motor to drive the pipetting unit to move on the guide rail (103).

4. the integrated nucleic acid detecting device according to claim 3, wherein: the pipetting unit comprises a connecting part (112) and a pipetting part (111), wherein the connecting part (112) has a structure that the guide rails (103) are sleeved and matched with each other, and the connecting part (112) is fixedly connected with one end of the pipetting part (111).

5. An integrated nucleic acid detecting device according to claim 1 or 2, wherein: the reagent storage unit is provided with a plurality of reagent storage holes (110).

6. The integrated nucleic acid detecting device according to claim 5, wherein: the rotating disc (108) comprises a rotating shaft (105) and a disc surface, a detection pore channel (109) for loading solution is arranged on the disc surface, and the rotating shaft (105) drives the disc surface to rotate.

7. a method for detecting a nucleic acid, comprising: the method adopts the integrated nucleic acid detection device of any one of claims 1 to 6 for detection, and comprises the following specific steps:

1) Adding a nucleic acid sample to a pore channel of a reagent storage unit, wherein other pore channels of the reagent storage unit are preloaded with a nucleic acid reaction reagent, and a nucleic acid detection reagent is encapsulated in the pore channel of the rotating disc (108);

2) The pipetting unit sucks a nucleic acid sample, the nucleic acid sample is sequentially mixed and reacted with a nucleic acid reaction reagent in the reagent storage unit by moving along a guide rail (103), the nucleic acid sample is matched with the rotating disc (108) to rotate, and the solution after reaction is subpackaged into pore channels of the rotating disc (108) by the pipetting unit for reaction to obtain a solution to be detected;

3) The solution to be detected is aligned to the detection hole (106) for detection through the rotation of the rotating disc (108).

8. the method for detecting a nucleic acid according to claim 7, wherein: the reagent storage unit in the step 1) is preloaded with binding solution, lysis solution, cleaning solution and mineral oil, and in the step 2), the reacted solution is subpackaged into pore channels of the rotating disc (108), and then the mineral oil is sequentially added.

9. The method for detecting a nucleic acid according to claim 8, wherein: the nucleic acid sample includes a microbial sample containing bacteria, viruses, or fungi.

Technical Field

The invention belongs to the technical field of nucleic acid detection devices, and particularly relates to an integrated nucleic acid detection device and an application method thereof.

Background

The conventional microbial nucleic acid detection process comprises three steps of sample pretreatment (nucleic acid extraction), nucleic acid amplification and nucleic acid detection. Extracting nucleic acid; firstly, extracting sample nucleic acid for nucleic acid detection; common methods include ethanol precipitation, silica gel column, glass bead, and magnetic bead; the basic principle of all the methods is four steps of sample lysis, combination, washing, elution and the like. The entire process for a single sample can be from about 40 minutes to several hours and sometimes exposed to toxic substances. Amplifying nucleic acid; common methods include temperature-variable amplification (such as PCR) and isothermal amplification (such as LAMP), which are both nucleic acid in vitro amplification techniques; PCR amplification refers to a process of in vitro copying daughter strand DNA complementary to the mother strand template DNA by taking the mother strand DNA as a template and a specific primer as an extension starting point under the catalysis of DNA polymerase through the steps of denaturation, annealing, extension and the like; the isothermal amplification reaction process is always maintained at a constant temperature, and the aim of rapid nucleic acid amplification is fulfilled by adding enzymes with different activities and respective specific primers. Detecting nucleic acid; agarose gel/polyacrylamide gel electrophoresis mode, including the processes of preparing gel, adding sample, electrophoresis, detection, etc.; detection by sequencing and expression means; qualitative or quantitative detection of the amplification product by a fluorescent dye or turbidity change; the operation steps are time-consuming and labor-consuming and have high cost.

All test links from an original sample to final detection are completed, the whole process needs 5-8 hours and is even longer, and test tubes need to be marked in each test link so as to avoid sample mismatching, so that the labor and the time are wasted; operators can easily contact toxic reagents, the reagent tube is easily polluted by the environment when the reagent tube is frequently opened and closed, and meanwhile, the reagent box and products can also pollute the environment; therefore, related supporting facilities need to be configured to ensure a pure experimental environment. In addition, the detection of multiple indices is more labor intensive.

Aiming at the complex test process, instruments such as a nucleic acid extractor, a nucleic acid amplifier, a real-time fluorescence quantitative instrument, a loop-mediated isothermal amplifier and the like are arranged on the market in each step to replace manual operation; at present, a liquid transfer workstation has high automation degree, comprises nucleic acid pretreatment, reaction configuration, temperature control, uniform mixing and the like, required matching reagents are placed on a workstation, and a program is set, so that the whole process of nucleic acid extraction and nucleic acid amplification can be completed. Because the liquid-transfering workstation is an open platform, in the whole operation process of many tests, an operator needs to irregularly seal the membrane of the 96-pore plate, open the mold or irregularly open and close a large number of tube covers, although a part of work is finished by the workstation, the whole process still needs a large amount of manual intervention, and the unattended operation of the whole process cannot be realized.

through retrieval, related automatic nucleic acid detecting devices have been disclosed, for example, the application with chinese patent application No. 201620190468.6 and publication date 2016.08.10 discloses a full-automatic nucleic acid detecting device, which comprises an upper cover and a lower cover connected with each other in the above-below order, wherein the upper side of the upper cover and the lower side of the lower cover are respectively provided with a fixing mechanism; the lower cover is internally provided with a sample injector with an upward opening, and the upper cover is provided with a jack which penetrates through the upper cover from top to bottom relative to the sample injector; the fixing mechanism of the upper cover is a triangular groove arranged in the center of the upper surface of the upper cover, and the fixing mechanism of the lower cover is a clamping groove arranged at the lower end of the side wall of the lower cover. When the device is used, the upper cover 1 is fixed with the rotating shaft through the triangular groove, and then a preset needle head in a needle head bottle is taken out and is arranged in an external assembly with a corresponding specification. The lower cover can be equipped with various sample applicators for nucleic acid extraction and PCR. The mouth of the reagent tube is provided with a silica gel plug, in the production process, the reagent tube is covered with the silica gel plug after being added with the reagent, and then the whole reagent tube can be inserted into the reserved round hole of the lower cover for fixation according to a certain sequence. After the upper cover and the lower cover are combined, the upper cover is screwed into the track combined with the lower cover. Through the rotation of operating the lower cover, make required injector reach the syringe needle below, the work of arranging is got to all kinds of reagents to rethread external component completion. Although the device can eliminate human errors in operation to a certain extent, improve the accuracy of reaction results and reduce the working strength and danger of operators, the device does not improve the detection speed to some extent.

Based on the defects in the prior art, a device which is simple in structure, high in detection speed, low in cost and capable of realizing integrated detection is urgently needed to be invented.

Disclosure of Invention

1. Technical problem to be solved

Aiming at the defects of complex equipment, high detection cost and easy cross contamination in nucleic acid detection in the prior art, the invention provides the integrated nucleic acid detection device and the integrated nucleic acid detection method which are simple and easy to operate, and the integrated nucleic acid detection device and the integrated nucleic acid detection method can realize full automation of nucleic acid detection including extraction, amplification and detection by combining a matched control system, and have the advantages of low detection cost, high detection speed and difficult sample pollution.

2. technical scheme

The invention provides an integrated nucleic acid detection device which comprises a shell, a reagent storage unit, a pipetting unit, a guide rail, a rotating disc and a detection hole arranged on the shell, wherein the guide rail penetrates through the shell in the axial direction, the reagent storage unit and the rotating disc are positioned below the guide rail, and the pipetting unit transfers liquid between the reagent storage unit and the rotating disc through moving along the guide rail.

The positions of the guide rails correspond to the arrangement positions of the reagent storage unit and the rotary disk, so that the pipetting unit can accurately correspond to liquid storage channels arranged in the reagent storage unit and the rotary disk.

The device firstly moves along a guide rail and moves up and down in the vertical direction through a liquid transfer unit, sucks and transfers a reagent pre-loaded in a reagent storage unit, and makes a nucleic acid sample to be detected and the pre-loaded reagent perform a series of reactions in the reagent storage unit; then, the liquid transfer unit is matched with the rotation of the rotating disc in a rotating mode, the solution after reaction is subpackaged to the pore channels in the rotating disc, the solution after reaction reacts with reagents pre-installed in the rotating disc to complete PCR amplification and detection reaction, the rotating disc is matched to rotate to enable the solution to be detected to sequentially correspond to the detection holes, and a detector is used for detecting.

as a further improvement of the invention, the device also comprises a liquid transfer pump and a gas guide tube which are respectively connected with the liquid transfer unit and the liquid transfer pump. The liquid transfer pump provides power for the liquid transfer unit to suck and release liquid.

As a further improvement of the invention, the guide rail is a ball screw, a sealing ring is sleeved on the guide rail to keep sealing, the pipetting unit is sleeved on the guide rail, the guide rail is connected with a motor, and the motor drives the screw to rotate so as to drive the pipetting unit to move on the guide rail.

As a further improvement of the invention, the pipetting unit comprises a connecting part and a pipetting part, wherein the connecting part has a structure that the guide rails are sleeved and matched with each other, the connecting part is fixedly connected with one end of the pipetting part, and the other end of the pipetting part is used for transferring liquid.

The device needs to be used with the automatic device of nucleic acid detection in a matched manner, the full automation of nucleic acid detection is realized, the module matched with the device comprises a temperature control module, an external power module and a blending module, the temperature control module is used for enabling nucleic acid amplification to keep a certain temperature, and the external power module comprises a power module which drives a liquid transfer unit to move up and down in the vertical direction. The external power module is matched with the spring to deform through magnetic force provided by the permanent magnet, and pushes the pipetting unit to move up and down in the vertical direction.

as a further improvement of the invention, a plurality of cylinders are arranged in the reagent storage unit, and reagent storage holes are arranged in the cylinders. The reagent storage unit is a trough body, and the trough body can be round, square or rectangular or in other shapes.

As a further development of the invention, the device further comprises a detection unit for the detection of the liquid in the rotating disc.

as a further improvement of the invention, the rotating disc comprises a rotating shaft and a disc surface, wherein a detection pore channel for loading solution is arranged on the disc surface, the rotating shaft drives the disc surface to rotate, and the rotating shaft is driven to rotate by a motor. The detection channel may be directly loaded with a solution or loaded with a solution by inserting a reaction tube.

As a further improvement, the invention provides a nucleic acid detection method, which uses the integrated nucleic acid detection device for detection and comprises the following specific steps:

1) Adding a nucleic acid sample into a pore channel of a reagent storage unit, wherein other pore channels of the reagent storage unit are preloaded with a nucleic acid reaction reagent, and a nucleic acid detection reagent is packaged in a detection pore channel of the rotating disc;

2) The pipetting unit sucks a nucleic acid sample, the nucleic acid sample is sequentially mixed and reacted with a nucleic acid reaction reagent in the reagent storage unit by moving along the guide rail, the nucleic acid sample is matched with the rotating disc to rotate, and the solution after reaction is subpackaged into a detection pore channel of the rotating disc by the pipetting unit for reaction to obtain a solution to be detected;

3) And (4) aligning the solution to be detected to the detection hole by rotating the rotating disc to detect.

As a further improvement of the invention, in the step 1), the reagent storage unit is pre-filled with binding solution, lysis solution, cleaning solution and mineral oil in sequence, and in the step 2), the reacted solution is subpackaged into pore channels of the rotating disc, and then the mineral oil is added in sequence.

as a further development of the invention, the nucleic acid sample comprises a microbial sample containing bacteria, viruses or fungi.

as a further improvement of the invention, the nucleic acid detection reagent comprises a fluorescent PCR reaction solution of a Texas Red probe.

3. Advantageous effects

(1) According to the integrated nucleic acid detection device, the whole reaction area is positioned in the shell, the arranged guide rail penetrates along the axial direction of the shell, the reagent storage unit and the rotary disk are positioned below the guide rail, liquid transfer is carried out by controlling the movement of the liquid transfer unit on the guide rail and the vertical up-and-down movement of the liquid transfer unit, sub-packaging can be rapidly completed by the rotation of the rotary disk, fluorescent detection can be carried out while sub-packaging reaction is carried out, the whole detection time is greatly shortened, the whole structure is simple, the size is small, instruments such as a nucleic acid extraction instrument, a liquid transfer workstation and the like do not need to be matched, the whole process of denaturation, annealing and extension in nucleic acid amplification can be automatically completed without personnel participation in the whole process, time and labor are saved, the convenience in detection is greatly.

(2) According to the integrated nucleic acid detection device, the reagent storage unit and the pore channels in the rotating disc can be loaded with the reaction solution in a manner of inserting the reaction test tube into the pore channels, so that the reaction test tube is discarded in time after the reaction is finished, and no cross contamination is generated.

Drawings

FIG. 1 is a top view of a nucleic acid detecting device of the present invention;

FIG. 2 is a front view of a nucleic acid detecting device of the present invention;

Wherein: 101. an air duct; 102. a liquid transferring pump; 103. a guide rail; 104. a housing; 105. a rotating shaft; 106. a detection hole; 107. a detector; 108. rotating the disc; 109. detecting a pore channel; 110. a reagent storage well; 111. a pipetting means; 112. and a connecting member.

Detailed Description

The following specific examples are provided to further illustrate the methods and embodiments of the present invention, but are not to be construed as limiting the invention.