KR20220096444A - Interface module for rt-pcr and apparatus using the same - Google Patents

Abstract

The present invention relates to an interface tube module for RT-PCR and a device using the same, and more particularly, to an interface tube module for RT-PCR in which a flexible membrane valve of the interface tube module for RT-PCR is mounted to increase the accuracy of sample movement.

Description

INTERFACE MODULE FOR RT-PCR AND APPARATUS USING THE SAME

The present invention relates to an interface tube module for RT-PCR and an apparatus using the same, and more particularly, to an interface tube module for RT-PCR that increases the accuracy of sample movement by mounting a flexible membrane valve of the interface tube module for RT-PCR and to a device using the same.

Polymerase Chain Reaction (PCR) is a molecular biological technology that replicates and amplifies a desired part of DNA. This technique can selectively amplify only specific DNA fragments of interest in a sample. In addition, the time required for amplification is short, and since it has high accuracy and can measure a small amount of DNA, it plays an important role in various fields such as molecular biology, medicine, criminal investigation, and classification of living things.

As a tool for confirming infectious diseases, RT-PCR (Real time PCR) has become a universal technology. As a screening device, the RT-PCR device has a stagnant level of technology through the development and optimization of bio-related technology, and equipment development by latecomers continues.

Most of the RT-PCR equipment is being operated by a professional inspection institution, and the technology is concentrating on processing a large number of samples at the same time.

However, the difficulty of using the RT-PCR device for on-site diagnosis is that extraction and fixation for extracting target DNA or RNA from a sample is performed using a separate automated equipment or manual operation performed by skilled workers. can't be done easily Also, since most automated equipment is designed to process many samples at the same time, it is difficult to apply when the number of samples to be processed at one time is small, such as in a primary medical institution.

Therefore, the entire process from DNA extraction process to real-time PCR is carried out in one step and development of an executable device without the intervention of skilled personnel is required. The cost is high, the on-site diagnosis is difficult, the measurement time and cost are high, so it is burdensome for general consumers to use it frequently, and it has problems in that it is difficult to quickly confirm the measurement result because samples must be collected and processed.

Accordingly, there is a need for research and development of an interface module to efficiently perform the entire process from DNA extraction to real-time PCR.

Korean Patent Laid-Open Publication No. 10-2012-0031188

The present invention was conceived to solve the above problems, and by mounting a flexible membrane valve on the interface tube module for RT-PCR, increases diagnostic efficiency, reduces contamination due to sample measurement, increases diagnostic convenience, and An object of the present invention is to provide an interface tube module for RT-PCR that can measure the entire process from preparation to measurement in an all-in-one manner, and an RT-PCR device having the same.

The interface tube module for RT-PCR according to an embodiment of the present invention includes an interface tube body; a hole formed at one side of the interface tube body; and a valve for opening and closing the hole.

It further includes; a connection lake for connecting one side of the interface tube body and the PCR tube, the one side of the interface tube body and the PCR connection lake are connected to the connection part, and the valve is located inside the connection part.

The valve is a flat membrane, is attached to the outside of the interface tube body to open and close the hole, and an upper part of the valve is attached to a region of the interface tube body located above the hole It opens and closes in one way direction.

The device according to an embodiment of the present invention includes an extraction tube module; an interface tube module located under the extraction tube module; and a PCR tube module located under the interface tube module, wherein the interface tube module is an interface tube module for RT-PCR.

The solution in the elution tube of the extraction tube module is moved into the interface tube body of the interface tube module by a lake connected to the end of the elution tube, and the solution in the interface tube body of the interface tube module is a flexible connection lake Move to the PCR tube of the PCR tube module through

Further comprising a motor for rotating the interface tube module and the PCR tube module, a moving axis connected to the motor is fixed to the lower center of the interface tube module, and passes through the PCR tube module and is fixed The interface tube module and PCR tube module are rotated by the rotation of the moving shaft by the motor, and the moving shaft is movable up and down, and the interface tube body is pulled by pulling the lower central part of the interface tube body. It is used to expand and contract the interface tube body by pushing up the lower central portion of the interface tube body.

The apparatus of the present invention represents a measuring apparatus for RT-PCR.

According to the all-in-one RT-PCR apparatus having an interface tube module for RT-PCR of the present invention, the operator extracts a small volume of the DNA solution that has been previously extracted using a precise pipette. The DNA solution is collected, transferred to a microtube, labeled, and then transferred to a PCR tube, allowing rapid and accurate measurement without going through a rather complicated process.

In addition, according to the all-in-one RT-PCR apparatus having an interface tube module for RT-PCR of the present invention, only one sample can be injected, multiple targets can be measured simultaneously, and the accident of changing the sample can be completely prevented. . Handling of small amounts of DNA solution can be achieved by using the centrifugal force and the difference in pressure.

In addition, according to the all-in-one RT-PCR apparatus having an interface tube module for RT-PCR of the present invention, centrifugal force is used as a method to collect a small amount of DNA solution at the end of the tube, and when moving to the interface tube, A flexible membrane valve can be applied to properly move the DNA solution by applying pressure.

1 and 2 are schematic diagrams showing the RT-PCR apparatus of the present invention.
3 is a schematic diagram showing an RT-PCR apparatus obtained by combining an extraction tube module, an interface tube module, and a PCR tube module.
4 shows a top side view of an extraction tube module, an interface tube module and a PCR tube module.
Figure 5 shows an enlarged view of the RT-PCR apparatus and PCR tube of the present invention.
6 shows an enlarged view of the RT-PCR apparatus and the interface tube module of the present invention.
7 shows an enlarged view of a partial cross-section of the interface tube module of the present invention.
8 is a schematic diagram showing the driving principle of the RT-PCR apparatus of the present invention.
9 is a schematic diagram showing an RT-PCR apparatus of the present invention.
10 is a schematic diagram showing an optical measurement module of the RT-PCR apparatus of the present invention.
11 shows a PCR tube and an enlarged view of the RT-PCR apparatus of the present invention.
12 is a schematic diagram showing the optical measurement of the RT-PCR apparatus of the present invention.
13 is a schematic diagram showing the driving principle of the RT-PCR device of the present invention.

Hereinafter, an interface tube module for RT-PCR according to an exemplary embodiment and an apparatus having the same will be described in detail with reference to the accompanying drawings.

In the following drawings, the same reference numerals refer to the same components, and the size of each component in the drawings may be exaggerated for clarity and convenience of description. Meanwhile, the embodiments described below are merely exemplary, and various modifications are possible from these embodiments. Hereinafter, what is described as "upper" or "upper" may include not only directly on in contact but also on non-contacting. Terms such as first, second, etc. may be used to describe various elements, but the elements should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another. The singular expression includes the plural expression unless the context clearly dictates otherwise. Also, when a part "includes" a component, it means that other components may be further included, rather than excluding other components, unless otherwise stated. In addition, terms such as "...unit" and "module" described in the specification mean a unit that processes at least one function or operation.

1 and 2 are schematic diagrams showing the RT-PCR apparatus of the present invention. 1 shows an RT-PCR apparatus in which an extraction tube module 100, an interface tube module 200, and a PCR tube module 300 are integrally combined, and FIG. 2 is an extraction tube module 100, an interface tube module ( 200), the PCR tube module 300 is separated to indicate the RT-PCR device coupled to the optical measurement module 400 .

As shown in FIG. 1 , in the RT-PCR apparatus of the present invention, the extraction tube module 100 is connected to the rotating unit 30 at the lower portion of the rail 10 , and the interface tube module is located at the lower portion of the extraction tube module 100 . 200 is detachably connected, and the PCR tube module 300 is detachably connected to the lower portion of the interface tube module 200 . Here, the extraction tube module 100 , the interface tube module 200 and the PCR tube module 300 are rotatable by the same motor 20 . For example, the extraction tube module 100, the interface tube module 200, and the PCR tube module 300 are rotatable by the rotation of the rotating part 30 connected to the lower part of the rail and the moving shaft 280 connected to the motor. .

2, in the RT-PCR apparatus of the present invention, the PCR tube module 300 is separated and coupled to the optical measurement module 400, and the PCR tube module 300 is rotatable by the motor 20 . The optical measurement module 400 , the PCR tube module 300 , and the motor 20 may be controlled by the controller 420 .

The extraction tube of the extraction tube module 100 of the present invention may include a tube for lysis buffer, a tube for magnetic beads, a cleaning tube, and an elution tube. Here, the tube for Lysis Buffer can be treated with a solution containing a substance that dissolves the cell wall, and the tube for magnetic beads is a magnetic high-density particle that chemically does not break the cell wall. It uses a magnetic substance that breaks the cell wall. Wash Buffer tube goes through Lysis Buffer tube and Magnetic Bead tube. can be removed The elution tube can be demagnetized so that the DNA is separated from the beads and separated into free particles in the solution.

That is, if the extraction process of the present invention is described in detail, when a sample is injected into any one of the extraction tubes and started, the first to sixth tubes rotate while the magnetic pole is inserted through the rotational movement of the plastic pole. The cell wall is destroyed. For example, a lysis buffer is mixed and heated at 70°C to 90°C, a magnetic pole moves to the first tube by attaching a magnetic bead in the second tube, and a pole containing a magnet in the first tube is left and right With a rotational motion (4,000 rpm to 8,000 rpm), the beads collide with the cells in the solution and the cell wall is destroyed. Next, stop the rotation and insert the magnetic pole to attach the separated DNA fragment to the magnetic bead, the magnetic pole and the plastic tube move upward with the DNA fragment attached at the same time, and the magazine-type extraction tube module 100 is 60 A rotation of 1/6 of 360° moves the cleaning tube under the pole. The DNA-attached pole is inserted into the cleaning solution, and the plastic tube rotates to remove the acid and protein residues attached to the DNA. This cleaning process is repeated three times with three cleaning tubes. Finally, the DNA is separated from the magnetic bead by repeated rotation in the elution solution in the Elution Tube, and only the beads are recovered by inserting the magnetic pole again. Only the elution solution and DNA remain in the elution tube.

Figure 3 is a schematic diagram for showing the RT-PCR apparatus obtained by combining the extraction tube module 100, the interface tube module 200 and the PCR tube module 300.

3 , the extraction tube module 100 and the interface tube module 200 may be connected by a fixed pipe 110 . The solution in the elution tube of the extraction tube module may move to the interface tube body of the interface tube module 200 through the pipe 110 . In addition, the interface tube module 200 and the PCR tube module 300 may be connected by a connection hose 230 having a flexible material. For example, one end of the connecting hose may be connected to the side of the interface tube body, and one end of the connecting lake may be connected in the PCR tube. In this way, the solution in the interface tube module 200 can move to the PCR tube module 300 through the connection lake 230 .

Extraction tube module 100 of the present invention may have a circular magazine shape. The extraction tube insertion hole of the extraction tube module 100 may be formed to be spaced apart from the center of the extraction tube module 100 at equal intervals.

Connecting the extraction tube module 100 and the interface tube module 200 by a fixed pipe 110 is the interface tube module 200 by a moving shaft 280 that enables rotation and vertical movement. Because it is supported on the lower part of the, that is, the interface tube body, the extraction tube module 100 and the interface tube module 200 are coupled and rotated so that the extraction tube module 100 and the interface tube module 200 are connected to a fixed pipe ( 110) is preferably connected. Here, the fixed pipe may be formed as an end of the elution tube. The inside of the end of the elution tube is filled with paraffin, and the paraffin is melted by heating a coil wound on the outside of the end of the elution tube, so that the solution in the elution tube can be moved to the interface tube module 200 .

In addition, the interface tube module 200 and the PCR tube module 300 may be connected by a connection hose 230 having a flexible material. The moving shaft 280 may be fixed to pass through the PCR tube module 300 . Since the lower part of the interface tube module 200, that is, the lower part of the interface tube body, is fixed by the moving shaft 280, the interface tube module 200 can rotate appropriately by the rotation of the moving shaft 280, In addition, the interface tube module 200 may be expanded and compressed by the vertical movement of the moving shaft 280 . As described above, the interface tube module 200 preferably has a flexible material in order to be expanded and compressed. In addition, by using the connection hose 230 having a flexible material, the connection hose 230 can be flexibly connected to the PCR tube. The interface tube body and connection hose can be used without limitation as long as they are made of a flexible material such as polyethylene, polypropylene, or polyvinyl chloride, as long as they have flexibility such as plastic or rubber.

4 shows a top side view of the extraction tube module 100 , the interface tube module 200 and the PCR tube module 300 . As shown in FIG. 4 , a bearing 170 for rotation of the extraction tube is provided inside the extraction tube module 100 of the present invention, and a bearing 370 for rotation of the PCR tube is provided inside the PCR tube module 300 . do. The extraction tube and PCR tube insertion hole of the PCR tube module 300 inserted into the extraction tube insertion hole 180 of the extraction tube module 100 by the extraction tube rotation bearing 170 and the PCR tube rotation bearing 370 ( 380), the inserted PCR tube is effectively rotatable.

Figure 5 shows an enlarged view of the RT-PCR apparatus and PCR tube of the present invention.

As shown in Figure 5 (a), the PCR tube 310 used in the PCR tube module 300 has a reagent 320 therein. The solution of the interface module flows into the PCR tube through the connection lake 230 . Here, a stopper 330 for blocking the inlet of the PCR tube is provided. The stopper 330 may use a semi-hermetic material. For example, a cork can be used. When the stopper 330 is used as a hermetic material, it is preferable to use a semi- hermetic material because it is difficult to flow into the PCR tube even when pressure is applied to the solution of the interface module.

6 shows an enlarged view of the RT-PCR apparatus and the interface tube module 200 of the present invention. 6, the interface tube module 200 of the present invention includes an interface tube body 210; a hole 270 formed on one side of the interface tube body 210; and a membrane-type valve 220 that opens and closes the hole 270 . Here, the hole 270 and the valve 220 may be provided in plurality.

7 shows an enlarged view of a partial cross-section of the interface tube module 200 of the present invention. 7 (a) shows a state in which the valve 220 is open, and FIG. 7 (b) shows a state in which the valve 220 is closed.

The solution that has moved to the interface tube body 210 stays at the bottom of the interface tube body 210, and when the heater block loaded with the PCR tube is strongly rotated, the solution at the bottom of the interface tube body is transferred to the valve at the edge of the tube by centrifugal force. By pushing to open the valve 220 located on one side of the interface tube body 210, the solution in the interface tube body 210 passes through the connection part 240 and the connection lake 230 and moves to the PCR tube. Here, the solution moved to the interface tube body 210 means a DNA extraction solution. The solution remaining in the interface tube body 210 is moved to an individual PCR tube through centrifugal force. Here, when more than a certain centrifugal force is applied to the valve 220 located on one side of the interface tube body 210 , the valve 220 is opened, and when less than a certain centrifugal force is applied, the valve 220 is closed.

The valve 220 is a flat membrane, larger than the hole 270 , and is attached to the outside of the interface tube body 210 to open and close the hole 270 . In addition, an upper portion of the valve 220 may be attached to a region of the interface tube body 210 positioned above the hole 270 to be opened and closed in one way. The valve 220 can be replaced with various types of small backflow prevention valves.

8 and 13 are schematic diagrams showing the driving principle of the RT-PCR apparatus of the present invention. After the extraction process as described above, as shown in FIG. 8 ( a ), the solution moves from the elution tube to the interface tube body 210 . A hot wire coil 350 surrounding the end of the elution tube is provided at the lower portion of the elution tube, and paraffin is melted at the end of the elution tube so that the solution is movable from the elution tube to the interface tube body 210 . As shown in FIG. 8(b) , an operation of rapidly moving the moving shaft 280 fixed to the lower center of the interface tube body 210 downward and slowly moving it upward in order to expand the interface tube body 210 . may be repeated to move the solution from the elution tube to the interface tube body 210 . Here, the ratio of the speed at which the moving shaft 280 moves downward and the speed at which it moves upward is preferably 1:0.1 to 0.5. Within the above range, the solution is movable from the elution tube to the interface tube body 210 without backflow. As shown in FIG. 8(c) , the solution of the interface tube body 210 may be moved to the PCR tube by rotating the interface tube body 210 on the moving shaft 280 . Since the cork stopper 330 has porosity, the solution of the interface tube body 210 can be moved to the PCR tube. The solution injected into the PCR tube is reacted with the reagent 320 . As shown in FIG. 8( d ), after the injection of the solution injected into the PCR tube is completed, the cork stopper 330 may be opened to transport the PCR tube module 300 to the optical measurement module 400 .

9 is a schematic diagram showing an RT-PCR apparatus of the present invention. As shown in FIG. 9 , the interface tube body 210 may be placed on the interface tube support 290 to be fixed to the moving shaft 280 . A surface of the interface tube supporter 290 facing the interface tube body 210 may have a concave shape to surround the interface tube body 210 when the interface tube body 210 is expanded. By providing the interface tube support 290 as described above, the flexible interface tube body 210 can have a predetermined shape and can be expanded.

10 and 12 are schematic diagrams for showing the optical measurement module 400 of the RT-PCR apparatus of the present invention. As shown in FIG. 10 , the optical measuring module 400 is provided by placing the moving part 40 under the rail 10 , and the light emitted from the optical lamp 480 is provided in the optical measuring module 400 . It passes through the sealing cover 430 and is transmitted and reflected by the solution in the PCR tube fixed to the harness 440, and can be measured by light. Here, the sealing cover 430 should be made of a transparent material, and should have good light transmittance and no fluorescence reaction. The PCR tube mounted on the heat block can be covered by applying pressure. Light passes through the sealing cover 430 to measure the PCR reaction. The harness 440 may operate the heat transfer of the heat block by pressing the pogo pin 450 located under the optical measurement module 400 .

11 shows a PCR tube and an enlarged view of the RT-PCR apparatus of the present invention. As shown in FIG. 11 , six PCR tubes are connected to the harness 440 in the form of a disk. Each PCR tube is fitted into a heat block, and the heat block is connected to the motor 20 . Here, the PCR tube is equipped with a heat block and cycled through a first temperature region (94°C), a second temperature region (54°C), and a third temperature region (72°C). For the operation of the RT-PCR device of the present invention, the fluctuation rate and stabilization rate of the temperature are important, and the temperature can be changed by heating the heat block with a Peltier or coil and rotating cooling water or external air inside the heat block.

The operation of the heat block is bound by a harness 440 of six PCR tube discs, and when the PCR tube set connected by the harness 440 is mounted on the heat block, pins inside the heat block fix the PCR tube. Here, the heat block rotates once in a temperature range of 70°C to 80°C, for example, in a section of 76°C in 6 steps. In each step, the LED is turned on for 1 to 3 seconds, for example, 1.5 seconds, and the fluorescence is measured with the CIS 10 to 50 times, for example 30 times. An average value of the measured values excluding data outside the error range is determined as a fluorescence value. The heat block rotates and the upper light source and sensor 410 are fixed, so that only the z-axis rotates. Here, a sealing film made of an elastic material may be used.

The technical idea should not be interpreted limited to the above-described embodiment of the present invention. Various modifications can be made at the level of those skilled in the art without departing from the gist of the present invention as claimed in the claims. Accordingly, such improvements and modifications fall within the protection scope of the present invention as long as it is apparent to those skilled in the art.

10 rail
20 motor
30 turns
40 moving part
100 Extraction Tube Modules
110 pipe
170 Bearing for rotating extraction tube
180 extraction tube inlet
200 interface tube module
210 interface tube body
220 valve
230 connecting lake
240 connections
270 holes
280 moving axis
290 interface tube support
300 PCR tube module
310 PCR tubes
320 reagent
330 plug
350 coils
Bearing for 370 PCR tube rotation
380 PCR tube insert
400 Optical Measurement Modules
410 sensor
420 controller
430 sealing cover
440 harness
450 Pocopin
460 heat blocks

Claims (6)

interface tube body;
a hole formed at one side of the interface tube body; and
An interface tube module for RT-PCR comprising a valve for opening and closing the hole. The method according to claim 1,
It further includes; a connection lake connecting one side of the interface tube body and the PCR tube,
The interface tube module for RT-PCR, characterized in that one side of the interface tube body and the PCR connection hose are connected to the connection part, and the valve is located inside the connection part. The method according to claim 1,
The valve is a flat membrane and is attached to the outside of the interface tube body to open and close the hole,
In addition, the interface tube module for RT-PCR, characterized in that the upper part of the valve is attached to a region of the interface tube body positioned above the hole to open and close in one way. extraction tube module;
an interface tube module located under the extraction tube module; and
A device comprising a; PCR tube module located below the interface tube module,
The interface tube module is an interface tube module for RT-PCR according to any one of claims 1 to 3. 5. The method according to claim 4,
The solution in the elution tube of the extraction tube module moves into the interface tube body of the interface tube module by a lake connected to the end of the elution tube,
The solution in the interface tube body of the interface tube module is characterized in that it moves to the PCR tube of the PCR tube module through a connection lake made of a flexible material. 5. The method according to claim 4,
Further comprising; a motor for rotating the interface tube module and the PCR tube module;
A moving axis connected to the motor is fixed to the lower center of the interface tube module, is formed to pass through the PCR tube module, is formed to be fixed to the PCR tube module, and rotation of the moving shaft by the motor The interface tube module and the PCR tube module are rotated by
In addition, the movable shaft is movable up and down, and the interface tube body is expanded by pulling the lower central portion of the interface tube body, and the interface tube body is contracted by pushing up the lower central portion of the interface tube body. device to do.

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