CN210856132U

CN210856132U - Integrated nucleic acid detection device

Info

Publication number: CN210856132U
Application number: CN201921374544.9U
Authority: CN
Inventors: 陈柱; 何农跃; 李松; 邓燕; 周枫林
Original assignee: Nanjing Arix Biotechnology Co ltd
Current assignee: Nanjing Arix Biotechnology Co ltd
Priority date: 2019-08-22
Filing date: 2019-08-22
Publication date: 2020-06-26
Anticipated expiration: 2029-08-22

Abstract

The utility model discloses an integration nucleic acid detection device, include casing, reagent storage unit, move liquid unit, guide rail, rotary disk and establish the detection hole on the casing, the guide rail runs through along the casing axial, reagent storage unit and rotary disk are located the below of guide rail, move liquid unit through shifting liquid between reagent storage unit and rotary disk along the guide rail motion. The utility model discloses a device is through moving liquid unit and accomplish a series of reactions at reagent storage unit along the guide rail motion, and rethread moves liquid unit and rotates mutually supporting along guide rail motion and rotary disk, with the inside pore of reaction back solution partial shipment to rotary disk, the reagent reaction of preassembleing in solution and the rotary disk accomplishes PCR amplification and detection reaction, and the rotary disk rotation of deuterogamying makes the solution that awaits measuring aim at the exploration hole, detects DNA or RNA amplification result, can accomplish degeneration, annealing, extension process in the nucleic acid amplification in the device through simple operation, has improved detection speed greatly.

Description

Integrated nucleic acid detection device

Technical Field

The utility model belongs to the technical field of nucleic acid detection device, specificly relate to an integration nucleic acid detection device.

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

To the defect that the equipment that nucleic acid detection among the prior art exists is complicated, detect with high costs, easy cross contamination, the utility model provides an integration nucleic acid detection device and method for simple easy operation combines supporting control system, can realize the full automatization including extraction, amplification, detection of nucleic acid detection, and detection cost is low, and detection speed is fast, and the sample is difficult for polluting.

2. Technical scheme

The utility model provides an integration nucleic acid detection device, include casing, reagent storage unit, move liquid unit, guide rail, rotary disk and establish the detection hole on the casing, the guide rail runs through along the casing axial, reagent storage unit and rotary disk are located the below of guide rail, move liquid unit through shifting liquid between reagent storage unit and rotary disk along the guide rail motion.

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 of the utility model firstly absorbs and transfers the reagent pre-installed in the reagent storage unit through the movement of the liquid transfer unit along the guide rail and the up-and-down movement in the vertical direction, and makes the nucleic acid sample to be detected and the pre-installed 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 utility model, the device still includes liquid-transfering pump and air duct, the air duct is connected with liquid-transfering unit and liquid-transfering pump respectively. The liquid transfer pump provides power for the liquid transfer unit to suck and release liquid.

As a further improvement, the guide rail be ball, the cover has the sealing washer to keep sealed on the guide rail, move liquid unit suit on the guide rail, the guide rail be connected with the motor, rotatory through the motor drive lead screw, the drive moves liquid unit and removes on the guide rail.

As a further improvement of the present invention, the pipetting unit comprises an adapting unit and a pipetting member, the adapting unit has a structure that is mutually matched with the guide rail suit, the adapting unit is fixedly connected with one end of the pipetting member, and the other end of the pipetting member 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, the reagent storage unit is internally provided with a plurality of cylinders, and the cylinders are internally provided with reagent storage holes. 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 improvement of the present invention, the apparatus further comprises a detection unit for detecting the liquid in the rotating disk.

As a further improvement, the rotary disk includes rotation axis and quotation, is equipped with the detection pore that is used for loading solution in the quotation that shows, the rotation axis drives the quotation rotatory, the rotation axis pass through motor drive rotation. The detection channel may be directly loaded with a solution or loaded with a solution by inserting a reaction tube.

As a further improvement of the present invention, the specific steps of using the device to perform detection are:

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 utility model, the reagent storage unit is preassembled in proper order in step 1) and is equipped with binding liquid, lysate, washing liquid, mineral oil, with reaction back solution partial shipment to the pore of rotary disk in step 2) back, add mineral oil again in proper order.

As a further improvement of the present invention, the nucleic acid sample comprises a microbial sample containing a bacterium, a virus or a fungus.

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

3. Advantageous effects

(1) The utility model discloses an integration nucleic acid detection device, holistic reaction zone is located inside the casing, the guide rail of setting runs through along the casing axial, reagent storage unit and rotary disk are located the below of guide rail, move liquid transfer through reciprocating of liquid unit motion and vertical direction on the guide rail through control, can accomplish the partial shipment rapidly through the rotation of rotary disk, because can carry out fluorescence detection when the partial shipment reaction goes on, holistic check-out time has been shortened greatly, the overall structure is simple, small, do not need supporting nucleic acid to draw the appearance, move liquid instruments such as workstation, and whole process need not personnel to participate in can accomplish the automatic degeneration in the nucleic acid amplification, annealing, the overall process of extension, time saving and labor saving, the convenience of detection has been improved greatly, the detection cost is reduced simultaneously.

(2) The utility model discloses an integration nucleic acid detection device, the pore in reagent memory cell and the rotary disk all can utilize and insert reaction test tube wherein mode load reaction solution, consequently will react the test tube and in time abandon, no cross contamination's production after the reaction is accomplished.

Drawings

FIG. 1 is a plan view of a nucleic acid detecting device according to the present invention;

FIG. 2 is a front view of a nucleic acid detecting device according to 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 further illustrative of the methods and solutions provided by the present invention, but should not be construed as limiting the invention.

Example 1:

the present embodiment provides an integrated nucleic acid detecting device, as shown in fig. 1, which includes a housing 104, a reagent storage unit, a pipetting unit, a guide rail 103, a rotary disk 108, and a detection hole 106 provided on the housing 104, the guide rail 103 penetrating in the axial direction of the housing 104, the reagent storage unit and the rotary disk 108 being located below the guide rail 103, and the pipetting unit transferring liquid between the reagent storage unit and the rotary disk 108 by moving along the guide rail 103.

As shown in fig. 2, the position of the guide rail 103 corresponds to the position where the reagent storage unit and the rotary disk 108 are provided, and ensures that the pipetting unit can correspond to the reagent storage unit below the pipetting unit and the liquid storage well provided in the rotary disk 108 when the pipetting unit moves along the guide rail.

The guide rail 103 in this embodiment is a lead screw, a seal ring is sleeved on the guide rail 103 to keep sealing, the pipetting unit has a matching structure sleeved on the guide rail 103, the guide rail 103 is connected with a motor, and the lead screw is driven to rotate by the motor to drive the pipetting unit to move on the guide rail 103.

The liquid transferring unit consists of a connecting part 112 and a liquid transferring part 111, wherein the connecting part 112 has a structure that the guide rail 103 is sleeved and matched with each other, the connecting part 112 is fixedly connected with one end of the liquid transferring part 111, and the connecting part 112 drives the liquid transferring part 111 to horizontally move along the guide rail 103.

The device still includes air duct 101 and liquid-transfering pump 102, air duct 101 is linked together with liquid-transfering pump 102 and liquid-transfering part 111 respectively, liquid-transfering part 111 on set up accurate scale, provide power through liquid-transfering pump 102 and absorb and release liquid, can also blow the liquid after mixing simultaneously and beat the mixing. It may also be operated with an external blending module.

The pipetting unit can also move up and down in the vertical direction, the power needs the cooperation of an external power module to provide, and the external power module cooperates with the spring deformation through the magnetic force provided by the permanent magnet to push the pipetting unit to move up and down in the vertical direction.

The reagent storage unit is a trough body, and the trough body can be round, square or rectangular or in other shapes. Set up a plurality of cylinders in the cell body, be provided with reagent storage hole 110 in the cylinder, can directly regard as the reaction tube with reagent storage hole 110, also can regard as the reaction tube with test tube disect insertion to reagent storage hole 110.

The rotating disc 108 comprises a rotating shaft 105, a motor connected to the rotating shaft 105 and a peripheral detection channel 109, the detection channel 109 being used for loading the solution. The detection channel 109 may be directly loaded with a solution or inserted into a test tube as a reaction tube.

The utility model discloses a still need cooperate outside temperature control module when the device uses, make the reaction system keep the uniform temperature, concrete working process as follows:

a) firstly, the pipetting unit moves along a guide rail 103, and a series of reactions are completed in a reagent storage unit;

b) the pipetting unit moves along the guide rail 103 and is matched with the rotation of the rotating disc 108, so that the reaction solution can be rapidly subpackaged to the detection pore channel 109 in the rotating disc 108, the reaction solution reacts with the reagent preinstalled in the rotating disc 108, the PCR amplification and detection reaction are completed, and the solution to be detected is obtained;

c) the rotation of the rotating disc 108 is coordinated to make the solution to be detected in each detecting channel 109 correspond to the detecting hole 106 in sequence, and the detecting hole 106 is aligned by the detector 107 (such as a photoelectric detector) for detection.

The apparatus of this example was used to detect nucleic acids from microorganisms in a sample. The microorganism can be bacteria, virus, fungi, etc. The rotating disc 108 comprises 20 pore channels, 20 microorganisms can be detected by respectively pre-filling LAMP reaction liquid of SYBR Green I fluorescent dye, the detector 107 adopts reflection type photoelectric detection and is used for detecting products of DNA or RNA, and the detection steps are as follows:

1) firstly, a pretreated sample is filled into the 1 st hole of a reagent storage unit through a liquid transfer unit, and the reagent storage unit is pre-filled with binding liquid/magnetic beads, lysis solution, cleaning solution I, cleaning solution II, eluent and mineral oil in sequence from the 2 nd hole;

2) the device is then placed in an automated nucleic acid detector. The detector is a device matched with the device. The pipetting unit starts to process the sample liquid according to a preset program under the driving of external equipment, the processes of binding solution, lysis solution, cleaning solution and the like are sequentially carried out, then the extracted nucleic acid is uniformly distributed to a plurality of detection pore channels 109 of the rotating disc 108, and finally mineral oil is added into the plurality of detection pore channels 109;

3) the whole detection process is carried out under the condition of constant temperature, and the temperature is about 60-65 ℃ and is provided by an external temperature control module. The detector emits blue light with a wavelength of 470nm, the blue light is focused on the solution to be detected of the detection pore channel 109 in the rotating disc 108 in a time-sharing manner, fluorescence with a wavelength of 520nm in the detection pore channel 109 is excited along with the progress of LAMP reaction, and finally, the photoelectric detector can detect the change of 20 peak signals. Whether the sample contains a certain microorganism is determined by the change of the photoelectric signal. Different LAMP reaction solutions detect different microorganisms.

Example 2

The nucleic acid detection of microorganisms, which may be bacteria, viruses, fungi, etc., is carried out using the apparatus of example 1. The method comprises the following specific steps:

1) firstly, a pretreated nucleic acid sample is filled into a 1 st hole of a reagent storage unit through a liquid transfer unit, and the reagent storage unit is pre-filled with binding liquid/magnetic beads, lysis solution, cleaning solution I, cleaning solution II, eluent and mineral oil in sequence from the 2 nd hole; the rotating disc 108 is internally provided with 20 detection channels 109 which are respectively pre-filled with fluorescent PCR reaction liquid of Texas Red probes to detect 20 microorganisms, and the detector adopts reflection-type photoelectric detection and is used for detecting products of DNA or RNA.

2) Under the drive of an external module, the pipetting unit processes sample liquid according to a preset program, extracts nucleic acid sequentially through processes of binding solution, lysis solution, cleaning solution and the like, then dispenses the extracted nucleic acid to the detection pore channels 109 of the rotating disc 108 in cooperation with the rotation of the rotating disc 108, finally adds mineral oil into the detection pore channels 109, and completes the processes of denaturation, annealing and extension in nucleic acid amplification in the detection pore channels 109 to obtain a solution to be detected;

3) the three temperature interval cycles of the whole detection process are controlled by an external temperature control module. Through the rotation of the rotating disc 108, the reaction solution to be detected sequentially corresponds to the detection holes 106, the detector emits green light with the wavelength of 565nm and focuses on the liquid to be detected in a time-sharing manner, fluorescence with the wavelength of 625nm in the detection hole channel 109 is excited along with the progress of PCR reaction, and finally, the photoelectric detector can detect the change of 20 peak signals. Whether the sample contains a certain microorganism is determined by the change of the photoelectric signal. Different PCR reactions detect different microorganisms.

The above description describes the preferred embodiments of the present invention, but the present invention is not limited to the details of the above embodiments, and the technical idea of the present invention can be modified to perform various equivalent transformations, which all belong to the protection scope of the present invention.

Claims

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 which is sleeved and matched with the guide rail (103), 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.