WO2021031227A1 - 一种一体化核酸检测器件及应用 - Google Patents

一种一体化核酸检测器件及应用 Download PDF

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WO2021031227A1
WO2021031227A1 PCT/CN2019/103042 CN2019103042W WO2021031227A1 WO 2021031227 A1 WO2021031227 A1 WO 2021031227A1 CN 2019103042 W CN2019103042 W CN 2019103042W WO 2021031227 A1 WO2021031227 A1 WO 2021031227A1
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nucleic acid
guide rail
rotating disk
reagent storage
pipetting
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PCT/CN2019/103042
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English (en)
French (fr)
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陈柱
何农跃
李松
邓燕
周枫林
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南京中大生物信息研究院有限公司
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Publication of WO2021031227A1 publication Critical patent/WO2021031227A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00584—Control arrangements for automatic analysers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/025—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations having a carousel or turntable for reaction cells or cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G01N2035/0439—Rotary sample carriers, i.e. carousels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G01N2035/0474—Details of actuating means for conveyors or pipettes

Definitions

  • the invention belongs to the technical field of nucleic acid detection devices, and specifically relates to an integrated nucleic acid detection device and an application method.
  • the conventional microbial nucleic acid detection process includes three steps: sample pretreatment (nucleic acid extraction), nucleic acid amplification, and nucleic acid detection.
  • Nucleic acid extraction nucleic acid detection first requires sample nucleic acid extraction; common methods include ethanol precipitation, silica gel column method, glass bead method, and magnetic bead method; the basic principles of all methods are four steps: sample lysis, binding, washing, and elution .
  • sample pretreatment nucleic acid extraction
  • nucleic acid detection first requires sample nucleic acid extraction
  • common methods include ethanol precipitation, silica gel column method, glass bead method, and magnetic bead method
  • the basic principles of all methods are four steps: sample lysis, binding, washing, and elution .
  • the entire process of a single sample takes about 40 minutes to several hours, and sometimes it is exposed to toxic substances.
  • Nucleic acid amplification commonly used methods include variable temperature amplification (such as PCR) and isothermal amplification (such as LAMP), both of which are nucleic acid in vitro amplification techniques;
  • PCR amplification refers to the use of parent strand DNA under the catalysis of DNA polymerase Template, with a specific primer as the starting point for extension, through denaturation, annealing, extension and other steps, the process of replicating the daughter strand DNA complementary to the parent strand template DNA in vitro; the isothermal amplification reaction process is always maintained at a constant temperature, by adding different Active enzymes and respective specific primers to achieve the purpose of rapid nucleic acid amplification.
  • Nucleic acid detection Nucleic acid detection; agarose gel/polyacrylamide gel electrophoresis method, including gel preparation, sample loading and electrophoresis, detection and other processes; detection by sequencing and expression methods; qualitative analysis of amplified products by fluorescent dyes or turbidity changes Or quantitative detection; its operation steps are time-consuming, labor-intensive, and costly.
  • test tube needs to be marked to avoid mistakes in the sample, which is laborious and laborious; the operators are easy to touch the toxic reagents and frequently Opening and closing the reagent tube is easy to be polluted by the environment, and at the same time, the reagent kit and products will also pollute the environment; therefore, it is necessary to configure related supporting facilities to ensure a pure experimental environment. In addition, it is more work to detect multiple indicators.
  • nucleic acid extraction instrument for replacing manual operations at each step, such as nucleic acid extraction instrument, nucleic acid amplification instrument, real-time fluorescence quantitative instrument, loop-mediated isothermal amplification instrument, etc.
  • the pipetting workstation is automated Higher, including nucleic acid pre-processing, reaction configuration, temperature control, mixing, etc. Place the required supporting reagents on the workstation and set the program to complete the whole process of nucleic acid extraction and nucleic acid amplification.
  • the pipetting workstation is an open platform, during the entire multi-test operation, the operator needs to perform the 96-well plate sealing and mold opening from time to time, or a large number of opening and closing work on the tube cover from time to time, although part of the work is done by the workstation , But the whole process still needs a lot of manual intervention, and the whole process cannot be unattended.
  • the Chinese patent application number 201620190468.6 and the publication date of 2016.08.10 discloses a fully automatic nucleic acid detection device, including the upper cover connected in the order of the above And the lower cover, the upper side of the upper cover and the lower side of the lower cover are respectively provided with fixing mechanisms; the lower cover is provided with a sampler with an upward opening, and the upper cover is provided with an upper side relative to the sampler.
  • the fixing mechanism of the upper cover is a triangular groove provided at the center of the upper surface of the upper cover, and the fixing mechanism of the lower cover is a card groove provided at the lower end of the side wall of the lower cover.
  • the upper cover 1 When the device is in use, the upper cover 1 is fixed through the triangular groove and the rotating shaft, and then the needles preset in the needle bottle are taken out and installed in the external components of the corresponding specifications.
  • the lower cover can be equipped with various sample injectors required for nucleic acid extraction and PCR.
  • the reagent tube is filled with the silica gel plug after adding the reagent, and then the entire reagent tube can be inserted into the round hole reserved in the lower cover in a certain order and fixed. After the upper cover and the lower cover are combined, screw the upper cover into the orbit where the lower cover is combined.
  • the device By manipulating the rotation of the lower cover, the required sample applicator reaches below the needle, and the external components are used to complete the removal and arrangement of various reagents.
  • the device can eliminate human errors in operation to a certain extent, improve the accuracy of response results, and reduce the work intensity and danger of operators, the device has not improved the detection speed.
  • the present invention provides an integrated nucleic acid detection device and method that is simple and easy to operate, combined with a matching control system, to achieve Nucleic acid detection includes full automation of extraction, amplification, and detection.
  • the detection cost is low, the detection speed is fast, and the sample is not easily contaminated.
  • the present invention provides an integrated nucleic acid detection device, comprising a casing, a reagent storage unit, a pipetting unit, a guide rail, a rotating disk and a detection hole provided on the casing, the guide rail penetrates along the axial direction of the casing, and The reagent storage unit and the rotating disk are located below the guide rail, and the liquid pipetting unit transfers liquid between the reagent storage unit and the rotating disk by moving along the guide rail.
  • the position of the guide rail corresponds to the installation position of the reagent storage unit and the rotating disk, ensuring that the pipetting unit can accurately correspond to the liquid storage channel provided in the reagent storage unit and the rotating disk.
  • the device of the present invention first moves the pipetting unit along the guide rail and moves up and down in the vertical direction to suck and transfer the pre-installed reagent in the reagent storage unit, and make a series of reactions between the nucleic acid sample to be tested and the pre-installed reagent in the reagent storage unit ; Then, by cooperating with the rotation of the rotating disk, the pipetting unit dispenses the reaction solution into the channels inside the rotating disk, and the reaction solution reacts with the reagents pre-installed in the rotating disk to complete the PCR amplification and detection reaction, and then cooperate The rotating disk rotates so that the solution to be tested corresponds to the detection hole in sequence, and the detector is used for detection.
  • the device further includes a pipetting pump and an air pipe, and the air pipe is respectively connected with the pipetting unit and the pipetting pump.
  • the pipetting pump provides power for the pipetting unit to suck and release liquid.
  • the guide rail is a ball screw
  • a sealing ring is sleeved on the guide rail to keep the seal
  • 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 , To drive the pipetting unit to move on the rail.
  • the pipetting unit includes a connecting part and a pipetting part
  • the connecting part has a structure in which a guide rail is fitted with each other, the connecting part is fixedly connected to one end of the pipetting part, and the pipetting part The other end of the part is used to transfer liquid.
  • the device needs to be used in conjunction with nucleic acid detection automation devices to realize full automation of nucleic acid detection.
  • the modules adapted to the device include a temperature control module, an external power module, and a mixing module.
  • the temperature control module is used for nucleic acid amplification.
  • the external power module includes a power module that drives the liquid transfer unit to move up and down in a vertical direction.
  • the external power module uses the magnetic force provided by the permanent magnet to cooperate with the spring deformation to push the pipetting unit to move up and down in the vertical direction.
  • 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 tank, and the tank may be round, square, rectangular or other shapes.
  • the device further includes a detection unit for detecting the liquid in the rotating disk.
  • the rotating disk includes a rotating shaft and a disk surface.
  • the disk surface is provided with detection holes for loading the solution.
  • the rotating shaft drives the disk surface to rotate, and the rotating shaft is driven to rotate by a motor.
  • the detection channel can be loaded with the solution directly or by inserting a reaction tube.
  • the present invention provides a nucleic acid detection method, the method uses the integrated nucleic acid detection device for detection, and the specific steps are:
  • nucleic acid sample is added to the channel of the reagent storage unit, the other channels of the reagent storage unit are pre-installed with nucleic acid reaction reagents, and the detection channel of the rotating disk is encapsulated with nucleic acid detection reagents;
  • the pipetting unit sucks the nucleic acid sample, and sequentially mixes and reacts with the nucleic acid reaction reagents in the reagent storage unit by moving along the guide rail, and cooperates with the rotation of the rotating disk.
  • the pipetting unit dispenses the reaction solution into the detection channel of the rotating disk. Perform the reaction to obtain the test solution;
  • step 1) the reagent storage unit is pre-loaded with binding solution, lysis solution, cleaning solution, and mineral oil in sequence.
  • step 2) the reacted solution is divided into the pores of the rotating disk. , Then add mineral oil in turn.
  • the nucleic acid samples include microorganism samples containing bacteria, viruses or fungi.
  • the nucleic acid detection reagent includes a PCR reaction solution with Texas Red probe fluorescence.
  • the overall reaction area is located inside the housing, and the guide rail is arranged axially through the housing, the reagent storage unit and the rotating disk are located under the guide rail, and the pipetting unit is controlled on the guide rail. Movement and vertical movement up and down for liquid transfer.
  • the dispensing can be completed quickly by the rotation of the rotating disk. Since the dispensing reaction can be carried out at the same time fluorescence detection, the overall detection time is greatly shortened, the overall structure is simple, and the volume is small. There is no need for supporting nucleic acid extractors, pipetting workstations and other instruments, and the whole process can automatically complete the whole process of denaturation, annealing, and extension in nucleic acid amplification without human involvement. This saves time and effort, greatly improves the convenience of detection, and reduces The cost of testing.
  • the reagent storage unit and the channels in the rotating disk can both be loaded with the reaction solution by inserting the reaction tube into it, so the reaction tube is discarded in time after the reaction is completed, without crossover The generation of pollution.
  • Figure 1 is a top view of a nucleic acid detection device of the present invention
  • Figure 2 is a front view of a nucleic acid detection device of the present invention.
  • 101 air duct; 102, pipetting pump; 103, guide rail; 104, housing; 105, rotating shaft; 106, detection hole; 107, detector; 108, rotating disk; 109, detection channel; 110, reagent Storage hole; 111, pipetting part; 112, connecting part.
  • the device includes a housing 104, a reagent storage unit, a pipetting unit, a guide rail 103, a rotating disk 108, and a detection device arranged on the housing 104.
  • Hole 106, the guide rail 103 penetrates axially along the housing 104, the reagent storage unit and the rotating disk 108 are located below the guide rail 103, and the pipetting unit moves between the reagent storage unit and the rotating disk 108 by moving along the guide rail 103 Transfer liquid.
  • the position of the guide rail 103 corresponds to the setting position of the reagent storage unit and the rotating disk 108 to ensure that when the pipetting unit moves along the guide rail, it can interact with the reagent storage unit and the liquid set in the rotating disk 108 below it. Corresponding to the storage channels.
  • the guide rail 103 is a lead screw
  • the guide rail 103 is covered with a sealing ring to keep the seal
  • the pipetting unit has a matching structure sleeved on the guide rail 103
  • the guide rail 103 is connected with a motor and driven by the motor The lead screw rotates to drive the pipetting unit to move on the guide rail 103.
  • the pipetting unit is composed of a connecting part 112 and a pipetting part 111.
  • the connecting part 112 has a structure in which the guide rail 103 is sleeved and matched with each other.
  • the connecting part 112 is fixedly connected to one end of the pipetting part 111.
  • the connecting part 112 drives the liquid transfer component 111 to move horizontally along the guide rail 103.
  • the device also includes an air guide tube 101 and a pipetting pump 102.
  • the air guide tube 101 is respectively connected to the pipetting pump 102 and the pipetting component 111.
  • the pipetting component 111 is provided with a precise scale, and the pipetting pump 102 It provides power to suck and release the liquid, and at the same time, the mixed liquid can be blown and mixed evenly. It can also be operated with an external mixing module.
  • the pipetting unit can also move up and down in the vertical direction, and the power needs to be provided by the cooperation of an external power module.
  • the external power module uses the magnetic force provided by the permanent magnet to cooperate with the spring deformation to push the pipetting unit to move up and down in the vertical direction. .
  • the reagent storage unit is a tank, and the tank may be round, square, rectangular or other shapes.
  • a plurality of cylinders are arranged in the tank, and reagent storage holes 110 are arranged in the cylinders.
  • the reagent storage holes 110 can be directly used as reaction tubes, or a test tube can be directly inserted into the reagent storage holes 110 as reaction tubes.
  • the rotating disk 108 includes a rotating shaft 105, a motor connected to the rotating shaft 105, and a detection hole 109 on the outer periphery, and the detection hole 109 is used for loading a solution.
  • the detection channel 109 can be directly loaded with solution or inserted into a test tube as a reaction tube.
  • the pipetting unit moves along the guide rail 103 to complete a series of reactions in the reagent storage unit;
  • the device of this embodiment is used for nucleic acid detection of microorganisms in a sample.
  • the microorganisms can be bacteria, viruses, fungi, etc.
  • the rotating disk 108 includes 20 channels, which are pre-installed with SYBR Green I fluorescent dye LAMP reaction solution, which can detect 20 kinds of microorganisms.
  • the detector 107 adopts reflective photoelectric detection for the detection of DNA or RNA products. The detection steps Specifically:
  • the pre-processed sample is injected into the first hole of the reagent storage unit through the pipetting unit.
  • the reagent storage unit is pre-installed with binding solution/magnetic beads, lysis solution, Cleaning fluid I, cleaning fluid II, eluent, mineral oil;
  • the pipetting unit starts to process the sample liquid according to the preset program, and then goes through the binding solution, lysis solution, cleaning solution and other processes in sequence, and then evenly divides the extracted nucleic acid into the multiple detection channels of the rotating disk 108 109. Finally, add mineral oil into multiple detection channels 109;
  • the whole detection process is carried out under constant temperature conditions, and the temperature is about 60°C ⁇ 65°C provided by the external temperature control module.
  • the detector emits blue light with a wavelength of 470 nm and time-sharing focused on the solution to be tested in the detection channel 109 in the rotating disc 108.
  • the photodetector can 20 peak signal changes are detected. The change of photoelectric signal is used to determine whether the sample contains certain microorganisms. Different LAMP reaction solutions detect different microorganisms.
  • the device of Example 1 is used for microbial nucleic acid detection.
  • the microorganisms can be bacteria, viruses, fungi, and so on. Specific steps are as follows:
  • the pre-processed nucleic acid sample is injected into the first hole of the reagent storage unit through the pipetting unit, and the reagent storage unit is pre-installed with binding solution/magnetic beads and lysis solution in sequence starting from the second hole , Cleaning fluid I, cleaning fluid II, eluent, mineral oil; there are 20 detection channels 109 in the rotating disk 108, and the PCR reaction solution with the fluorescence of Texas Red probe is pre-installed, which can detect 20 kinds of microorganisms.
  • the detector adopts Reflective photoelectric detection is used for the detection of DNA or RNA products.
  • the pipetting unit processes the sample liquid according to a preset program, and then extracts nucleic acids through the binding solution, lysis solution, and cleaning solution in sequence, and then cooperates with the rotation of the rotating disk 108 to extract nucleic acids.
  • the nucleic acid is distributed into the detection channels 109 of the plurality of rotating disks 108, and finally mineral oil is added to the plurality of detection channels 109, and the denaturation, annealing, and extension processes in nucleic acid amplification are completed in the detection channels 109 to obtain the solution to be tested;
  • the three temperature interval cycles of the whole detection process are controlled by an external temperature control module.
  • the reaction solution to be tested corresponds to the detection hole 106 in turn.
  • the detector emits green light with a wavelength of 565 nm and time-sharing focused on the liquid to be tested.
  • the detection channel 109 with a wavelength of 625 nm is excited Fluorescence, and finally, the photodetector can detect 20 peak signal changes. The change of photoelectric signal is used to determine whether the sample contains certain microorganisms. Different PCR reaction solutions detect different microorganisms.

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Abstract

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。

Description

一种一体化核酸检测器件及应用 技术领域
本发明属于核酸检测器件技术领域，具体的涉及一种一体化核酸检测器件及应用方法。
背景技术
常规的微生物核酸检测过程包括样本前处理(核酸提取)、核酸扩增、核酸检测三个步骤。核酸提取；核酸检测首先要进行样本核酸提取；常见方法有乙醇沉淀法、硅胶柱法、玻璃珠法、磁珠法；所有方法基本原理都是裂解样本、结合、清洗、洗脱等四个步骤。单个样本整个过程约40分钟到数小时，有时会接触到有毒物质。核酸扩增；常用方法有变温扩增(如PCR)和等温扩增(如LAMP)，都是一种核酸体外扩增技术；PCR扩增是指在DNA聚合酶催化下，以母链DNA为模板，以特定引物为延伸起点，通过变性、退火、延伸等步骤，体外复制出与母链模板DNA互补的子链DNA的过程；等温扩增反应过程始终维持在恒定的温度下，通过添加不同活性的酶和各自特异性引物来达到快速核酸扩增的目的。核酸检测；琼脂糖凝胶/聚丙烯酰胺凝胶电泳方式，包括制胶、加样和电泳、检测等过程；通过测序和表达方式来检测；通过荧光染料或浊度变化来对扩增产物定性或定量检测；其操作步骤都是耗时耗力，成本高。
完成一个从原始样本到最终检测的所有试验环节，整个过程需要5～8小时，甚至更长，在每个试验环节都需要标记试管以免弄错样本，费事费力；操作人员容易接触有毒试剂，频繁开闭试剂管容易被环境污染，同时试剂盒和产物也会污染环境；因此，需要配置相关配套设施，保证纯净的实验环境。此外，多个指标检测其工作量更大。
针对上述复杂试验过程，每个步骤市场上有一些仪器替代人工操作，如核酸提取仪、核酸扩增仪、实时荧光定量仪、环介导等温扩增仪等；目前，移液工作站是自动化程度较高，包括核酸前处理、反应配置、温度控制、混匀等，将所需的配套试剂放置于工作站台上，设置好程序，可以完成核酸提取和核酸扩增全过程。由于移液工作站为开放平台，在整个多不试验操作过程中，操作者需要不定期进行96孔板封膜、开模或不定期进行大量的管盖开闭工作，虽然一部分工作由工作站来完成，但整个仍需要人工大量介入，无法实现整个过程的无人值守。
经检索，相关的自动化核酸检测器件已得到公开，如中国专利申请号为201620190468.6，公开日期为2016.08.10的申请案公开了一种全自动的核酸检测器件，包括以上下次序相互连接的上盖和下盖，所述上盖的上侧和下盖的下侧分别设置有固定机构；所述下盖内设置有开口向上的加样器，上盖上相对于该加样器设置有从上到下贯穿上盖的插孔；所述上盖的固定机构为上盖上表面中心位置设置的三角槽，所述下盖的固定机构为下盖侧壁下端位置设置的卡槽。该装置在使用时通过三角槽跟转轴进行上盖1的固定，然后取出针头瓶内预置的针头， 并装入相应规格的外部组件中。下盖中可装上装有核酸提取和PCR所需的各种加样器。试剂管的管口处有硅胶塞，在生产过程中，试剂管中加入试剂后盖上硅胶塞，然后整个试剂管可按一定的次序插入下盖预留的圆孔中固定。上盖、下盖相合后，将上盖旋与下盖相合的轨道中。通过操纵下盖的转动，使得所需加样器到达针头下方，再通过外部组件完成对各类试剂的取排工作。该装置虽然能够在可一定程度消除操作中的人为误差，提高反应结果的准确性，减少操作人员的工作强度及危险性，然而该装置并未在检测速度上有所提高。
基于现有技术中存在的缺陷，亟需发明一种构造简单，检测速度快，成本低，能够实现一体化检测的器件。
发明内容
1.要解决的技术问题
针对现有技术中的核酸检测存在的设备复杂、检测成本高、容易交叉污染的缺陷，本发明提供用于简单易操作的一种一体化核酸检测装置及方法，结合配套的控制系统，可实现核酸检测的包括提取、扩增、检测的全自动化，检测成本低，检测速度快，样品不易污染。
2.技术方案
本发明提供了一种一体化核酸检测器件，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。
所述导轨的位置与试剂存储单元和旋转盘的设置位置相互对应，保证移液单元能够与准确对应于试剂存储单元和旋转盘内设置的液体存放孔道。
本发明的装置首先通过移液单元沿导轨运动以及在竖直方向上下运动，吸取并转移试剂存储单元内预装的试剂，在试剂存储单元内使待测核酸样本与预装试剂进行一系列反应；然后，通过与旋转盘转动的配合，移液单元将反应后溶液分装至旋转盘内部的孔道，反应后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与探测孔依次对应，利用检测器进行检测。
作为本发明更进一步的改进，所述装置还包括移液泵和导气管，所述导气管分别与移液单元和移液泵连接。所述的移液泵为移液单元吸取和释放液体提供动力。
作为本发明更进一步的改进，所述的导轨为滚珠丝杠，导轨上套有密封圈保持密封，所述移液单元套装在导轨上，所述的导轨与电机连接，通过电机驱动丝杠旋转，带动移液单元在导轨上移动。
作为本发明更进一步的改进，所述移液单元包括连接部件和移液部件，所述连接部件具有导轨套装相互配合的结构，所述连接部件与移液部件的一端固定连接，所述移液部件另一端用于转移液体。
该装置需要与核酸检测自动化器件配套使用，实现核酸检测全自动化，所述与装置适配的模块包括温控模块、外部动力模块、混匀模块，所述的温控模块用于使核酸扩增保持一定温度，所述外部动力模块包括带动移液单元在竖直方向上下移动的动力模块。所述外部动力模块通过永久磁铁提供的磁力配合弹簧形变，推动移液单元在竖直方向上下运动。
作为本发明更进一步的改进，所述试剂存储单元内设置多个圆柱体，所述圆柱体内设置有试剂存储孔。所述试剂存储单元为槽体，所述槽体可以为圆形、方形或长方形或其他形状。
作为本发明更进一步的改进，所述装置还包括检测单元，所述检测单元用于旋转盘内液体的检测。
作为本发明更进一步的改进，所述旋转盘包括旋转轴与盘面，所示盘面上设有用于装载溶液的检测孔道，所述旋转轴带动盘面旋转，所述的旋转轴通过电机驱动旋转。所述检测孔道可以直接装载溶液或通过安插反应管来装载溶液。
作为本发明更进一步的改进，本发明提供了一种核酸检测方法，所述方法应用所述的一体化核酸检测装置进行检测，具体的步骤为：
1)将核酸样品加至试剂存储单元的孔道，所述试剂存储单元其他孔道预装有核酸反应试剂，所述旋转盘的检测孔道内封装有核酸检测试剂；
2)移液单元吸取核酸样品，通过沿导轨移动依次与试剂存储单元内的核酸反应试剂分别混合、反应，配合旋转盘旋转，移液单元将反应后溶液分装至旋转盘的检测孔道内，进行反应，得到待测溶液；
3)通过旋转盘旋转使待测溶液对准探测孔，进行检测。
作为本发明更进一步的改进，步骤1)中试剂存储单元依次预装有结合液、裂解液、清洗液、矿物油，所述步骤2)中将反应后溶液分装至旋转盘的孔道内后，再依次加入矿物油。
作为本发明更进一步的改进，所述核酸样本包括含有细菌、病毒或真菌的微生物样本。
作为本发明更进一步的改进，所述核酸检测试剂包括Texas Red探针荧光的PCR反应液。
3.有益效果
(1)本发明的一体化核酸检测器件，整体的反应区域位于壳体内部，设置的导轨沿壳体轴向贯穿，试剂存储单元和旋转盘位于导轨的下方，通过控制移液单元在导轨上运动以及竖直方向的上下移动进行液体转移，通过旋转盘的旋转可迅速完成分装，由于分装反应进行的同时可以进行荧光检测，大大缩短了整体的检测时间，整体构造简单，体积小，不需要配套核酸提取仪、移液工作站等仪器，且整个过程无需人员参与即可自动完成核酸扩增中变性、退火、延伸的全过程，省时省力，大大提高了检测的便利性，同时降低了检测成本。
(2)本发明的一体化核酸检测器件，所述试剂存储单元和旋转盘内的孔道均可以利用将反应试管插入其中的方式装载反应溶液，因此在反应完成后将反应试管及时丢弃，无交叉污染的产生。
附图说明
图1是本发明的一种核酸检测器件的俯视图；
图2是本发明的一种核酸检测器件的前视图；
其中：101、导气管；102、移液泵；103、导轨；104、壳体；105、旋转轴；106、探测孔；107、检测器；108、旋转盘；109、检测孔道；110、试剂存储孔；111、移液部件；112、连接部件。
具体实施方式
以下具体实施例是对本发明提供方法与技术方案的进一步说明，但不应理解成对本发明的限制。
实施例1：
本实施例提供了一种一体化核酸检测器件，如图1所示，所述器件包括壳体104、试剂存储单元、移液单元、导轨103、旋转盘108和设在壳体104上的探测孔106，所述导轨103沿壳体104轴向贯穿，所述试剂存储单元和旋转盘108位于导轨103的下方，所述移液单元通过沿导轨103运动在试剂存储单元和旋转盘108之间转移液体。
如图2所示，所述导轨103的位置与试剂存储单元和旋转盘108的设置位置相对应，保证移液单元沿导轨移动时，能够与其下部的试剂存储单元和旋转盘108内设置的液体存放孔道相对应。
本实施例中所述的导轨103为丝杠，导轨103上套有密封圈保持密封，所述移液单元具有套装在导轨103上的配合结构，所述的导轨103与电机连接，通过电机驱动丝杠旋转，带动移液单元在导轨103上移动。
所述移液单元由连接部件112和移液部件111组成，所述连接部件112具有导轨103套装相互配合的结构，所述连接部件112与移液部件111的一端固定连接，所述的连接部件112带动移液部件111在沿着导轨103水平移动。
所述装置还包括导气管101和移液泵102，所述导气管101分别与移液泵102和移液部件111相连通，所述的移液部件111上设置精确刻度，通过移液泵102提供动力来吸取和释放液体，同时还可以对混合后的液体进行吹打混匀。也可以利用外部的混匀模块来操作。
所述的移液单元还能够在竖直方向上下移动，该动力需要外部动力模块配合来提供，所述外部动力模块通过永久磁铁提供的磁力配合弹簧形变，推动移液单元在竖直方向上下运动。
所述试剂存储单元为槽体，所述槽体可以为圆形、方形或长方形或其他形状。所述槽体内设置多个圆柱体，所述圆柱体内设置有试剂存储孔110，可以直接以试剂存储孔110作为反应管，也可以将试管直接插入至试剂存储孔110作为反应管。
所述旋转盘108包括旋转轴105、与旋转轴105连接的电机和外周的检测孔道109，所述检测孔道109用于装载溶液。所述的检测孔道109可以直接装载溶液或插入试管作为反应管。
本发明的器件使用时还需要配合外部温控模块，使反应体系保持一定温度，具体工作过程如下：
a)首先移液单元沿导轨103运动，在试剂存储单元完成一系列反应；
b)移液单元沿导轨103运动与旋转盘108转动相互配合，可以快速的将反应溶液分装至旋转盘108内部的检测孔道109，所述反应溶液和旋转盘108内预装的试剂进行反应，完成PCR扩增及检测反应，得到待测溶液；
c)配合旋转盘108转动使每个检测孔道109内的待测溶液与探测孔106依次对应，利用检测器107(如光电检测器)对准探测孔106，进行检测。
利用本实施例的装置对样本中微生物进行核酸检测。微生物可以是细菌、病毒和真菌等。旋转盘108内包括20个孔道，分别预装SYBR Green I荧光染料的LAMP反应液，即可检测20种微生物，检测器107采用反射式光电检测，用于DNA或者RNA的产物的检测，检测步骤具体为：
1)首先将预处理好的样本通过移液单元加注到试剂存储单元的第1个孔，所述的试剂存储单元从第2个孔开始依次预装了结合液/磁珠、裂解液、清洗液I、清洗液II、洗脱液、矿物油；
2)然后将器件放置于核酸自动检测仪里面。该检测仪是与该装置配套的设备。在外部设备的驱动下移液单元开始按照预设程序对样本液体进行处理，依次经过结合液、裂解液、清洗液等过程，然后再将提取的核酸均分至旋转盘108的多个检测孔道109，最后将矿物油加入多个检测孔道109；
3)整个检测过程在恒温条件下进行，温度大约在60℃～65℃由外部的温控模块提供。检测器发出470nm波长的蓝光分时聚焦于旋转圆盘108内的检测孔道109的待测溶液，随着LAMP反应的进行，激发出检测孔道109内波长为520nm的荧光，最后，光电探测器可以检测到20个峰值信号的变化。通过光电信号的变化来确定样本中是否含有某种微生物。不同的LAMP反应液检测不同的微生物。
实施例2
利用实施例1的装置进行微生物核酸检测，微生物可以是细菌、病毒和真菌等。具体步骤如下：
1)首先将预处理好的核酸样本通过移液单元加注到试剂存储单元的第1个孔，所述的试剂存储单元从第2个孔开始依次预装了结合液/磁珠、裂解液、清洗液I、清洗液II、洗脱液、矿物油；旋转盘108内有20个检测孔道109，分别预装Texas Red探针荧光的PCR反应液，即可检测20种微生物，检测器采用反射式光电检测，用于DNA或者RNA的产物的检测。
2)在外部模块的驱动下，所述的移液单元按照预设程序对样本液体进行处理，依次经过结合液、裂解液、清洗液等过程提取核酸，然后配合旋转盘108的旋转将提取的核酸分装至 多个旋转盘108的检测孔道109，最后将矿物油加入多个检测孔道109，在检测孔道109内完成核酸扩增中变性、退火、延伸过程，得到待测溶液；
3)整个检测过程三个温度区间循环由外部的温控模块控制。通过旋转盘108旋转，待测反应溶液依次与探测孔106对应，检测器发出565nm波长的绿光分时聚焦于待测液体，随着PCR反应的进行，激发出检测孔道109内波长为625nm的荧光，最后，光电探测器可以检测到20个峰值信号的变化。通过光电信号的变化来确定样本中是否含有某种微生物。不同的PCR反应液检测不同的微生物。
以上描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种等同变换，这些等同变换均属于本发明的保护范围。

Claims (9)

  1. 一种一体化核酸检测器件，其特征在于：包括壳体(104)、试剂存储单元、移液单元、导轨(103)、旋转盘(108)和设在壳体(104)上的探测孔(106)，所述导轨(103)沿壳体(104)轴向贯穿，所述试剂存储单元和旋转盘(108)位于导轨(103)的下方，所述移液单元通过沿导轨(103)运动在试剂存储单元和旋转盘(108)之间转移液体。
  2. 根据权利要求1所述的一体化核酸检测器件，其特征在于：所述器件还包括移液泵(102)和导气管(101)，所述移液单元和移液泵(102)通过导气管(101)连接。
  3. 根据权利要求1或2所述的一体化核酸检测器件，其特征在于：所述器件还包括用以驱动导轨(103)旋转的电机，所述导轨(103)为丝杠，所述移液单元套装在导轨(103)上，通过电机驱动导轨(103)旋转，带动移液单元在导轨(103)上移动。
  4. 根据权利要求3所述的一种一体化核酸检测器件，其特征在于：所述移液单元包括连接部件(112)和移液部件(111)，所述连接部件(112)具有导轨(103)套装相互配合的结构，所述连接部件(112)与移液部件(111)的一端固定连接。
  5. 根据权利要求1或2所述的一种一体化核酸检测器件，其特征在于：所述试剂存储单元上设有多个试剂存储孔(110)。
  6. 根据权利要求5所述的一种一体化核酸检测器件，其特征在于：所述旋转盘(108)包括旋转轴(105)与盘面，所示盘面上设有用于装载溶液的检测孔道(109)，所述旋转轴(105)带动盘面旋转。
  7. 一种核酸检测方法，其特征在于：所述方法应用权利要求1～6任意一项所述的一体化核酸检测器件进行检测，具体的步骤为：
    1)将核酸样本加至试剂存储单元的孔道，所述试剂存储单元其他孔道预装有核酸反应试剂，所述旋转盘(108)的孔道内封装有核酸检测试剂；
    2)移液单元吸取核酸样品，通过沿导轨(103)移动依次与试剂存储单元内的核酸反应试剂分别混合、反应，配合旋转盘(108)旋转，移液单元将反应后溶液分装至旋转盘(108)的孔道内，进行反应，得到待测溶液；
    3)通过旋转盘(108)旋转使待测溶液对准探测孔(106)进行检测。
  8. 根据权利要求7所述的核酸检测方法，其特征在于：步骤1)中试剂存储单元预装有结合液、裂解液、清洗液、矿物油，所述步骤2)中将反应后溶液分装至旋转盘(108)的孔道内后，再依次加入矿物油。
  9. 根据权利要求8所述的核酸检测方法，其特征在于：所述核酸样本包括含有细菌、病毒或真菌的微生物样本。
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