WO2024117301A1 - Cartridge for gene extraction and detection - Google Patents

Abstract

The present invention relates to a cartridge for gene extraction and detection, wherein identification of bacteria or strains causing infectious diseases and multi-detection of antibiotic resistance genes are performed to enable early selection of antibiotics appropriate for a patient. The present invention provides a cartridge for gene extraction and detection, comprising: a first body including a plurality of side chambers arranged outside a central chamber thereof; the central chamber located in the central part of the first body; a lower reagent moving means configured to rotate at the bottom of the central chamber and connect the side chamber to the central chamber; and a pump connected to the upper part of the central chamber to pressurize or depressurize the interior of the central chamber.

Description

Cartridges for gene extraction and detection

The present invention relates to a cartridge for gene extraction and detection, and more specifically, to gene extraction and detection that enables early selection of an appropriate antibiotic for a patient by multiple detection of antibiotic resistance gene information and identification of bacteria or strains causing infectious diseases. It's about cartridges.

Sepsis is an infectious disease with a very high mortality rate, accounting for approximately 25-30% of intensive care unit patients, and one person dies from sepsis every three seconds. Rapid diagnosis is a very important field, with the survival rate decreasing by 9% for every hour that treatment is delayed, but accurate prescriptions can only be made after a long 3-5 day culture process and antibiotic susceptibility testing after any initial prescription. Initial prescriptions are completely ineffective for 42% of patients, and more antibiotics than necessary are prescribed to 21% of patients. Therefore, it is necessary to eliminate drug abuse that occurs during this process.

A variety of technologies are being used to diagnose sepsis and test antibiotic susceptibility. Most of the existing technologies are unable to directly separate (purify) bacteria and fungi that exist in very small amounts in the blood, so they require a culturing process for a certain amount of time. Many attempts are being made to reduce this incubation time, and among them, attempts are being made to isolate the causative bacteria of sepsis using technology to capture bacteria and fungi in the blood and multiplex gene amplification technology.

Among these, the technology for capturing bacteria and fungi in the blood is a method of capturing desired bacteria using specific proteins. In the present invention, apolipoprotein is used to isolate sepsis-causing bacteria. Apolipoprotein H (ApoH) or beta2-glycoprotein I is an acute phase protein circulating in human plasma. It has been shown that ApoH can bind with high affinity to lipopolysaccharide (LPS), a major component of the outer wall of Gram-negative bacteria, and to specific proteins of Gram-positive pathogens. The broad specificity of ApoH allows it to be used as a marker for capture of bacterial and fungal pathogens in blood samples. ApoH can be coated on magnetic beads, and capturing pathogens using these beads can also work on fungi. When magnetic beads are mixed with blood and react, if there is an infectious agent (bacteria causing sepsis) in the blood, only the infectious agent can attach to the magnetic beads. If the magnetic beads are separated with a magnet, the infectious agent in the blood can be separated and concentrated.

In addition, multiple gene amplification technology (Nested polymerase chain reaction, Nested PCR) is a modification of PCR and is a method to reduce non-specific amplification of non-specific base sequences other than the base sequence of the target gene. Conventional PCR requires a complementary primer at the end of the base sequence of the target gene, and PCR products are amplified according to the cycle, but Nested PCR binds specifically to the base sequence of the target gene to increase amplification efficiency. The set of primers is used in two consecutive PCRs. At this time, the first PCR amplification product may include amplification of a non-specific base sequence, but the first PCR amplification product can be used as a target gene for the second PCR. In addition, the second PCR can increase the amplification accuracy of the base sequence of the final target gene by using an inner primer that can be attached to the base sequence nested within the first PCR amplification product or by using the ‘hemi, semi-nesting’ method. In other words, nested PCR can be used to increase the accuracy and reliability of multiple gene amplification of multiple pathogens that can cause sepsis.

However, technology for capturing bacteria and fungi in the blood and multiplex gene amplification technology requires complex device configuration and work processes at each step. Accordingly, specialized workers with separate training are required, there is a risk of accidents due to leakage or sample contamination during the testing process, and there is a problem of requiring too much work time to perform the test at a busy diagnostic site. Therefore, there is a need for a new testing device that automates the isolation of these bacteria and amplification of genes to quickly identify the bacteria causing sepsis and at the same time quickly identifies antibiotic resistance genes to enable early selection of antibiotics suitable for the patient.

In order to solve the above-mentioned problems, the present invention seeks to provide a cartridge for gene extraction and detection that enables early selection of antibiotics appropriate for the patient by identifying the bacteria or strains causing infectious diseases and detecting multiple antibiotic resistance gene information. .

In order to solve the above-described problem, the present invention includes a first body in which a plurality of side chambers are arranged outside the central chamber; a central chamber located in the center of the first body; a lower reagent moving means that rotates at the bottom of the central chamber to connect the central chamber and the side chamber; and a pump connected to the upper part of the central chamber to pressurize or depressurize the interior of the central chamber.

In one embodiment, the plurality of side chambers are; an injection chamber into which the sample tube is coupled; A collection reagent chamber loaded with a solution for collecting bacteria; A magnetic bead chamber loaded with a magnetic bead light buffer solution; A cleaning chamber loaded with a cleaning solution; A nucleic acid extraction chamber loaded with a buffer for nucleic acid elution; And it may include a connection chamber with a PCR channel connected to the upper part.

In one embodiment, a lid that closes the upper part of the central chamber and the side chamber is coupled to the upper part of the first body, and a pump connection portion to which the pump is connected may be formed in the center of the lid.

In one embodiment, one side of the PCR channel is connected to one side of the lid, and the bottom of one side of the PCR channel may be in communication with the connection chamber.

In one embodiment, the lower reagent moving means includes a moving channel therein, one side of which is fixed to the center of the central chamber, and the other side is selectively moved according to the rotation of the lower reagent moving means. It can be connected to two side chambers.

In one embodiment, the lower reagent moving means may move the sample, reagent, and magnetic beads between the central chamber and the side chamber through the pressure of the central chamber.

In one embodiment, a second body is coupled to the lower part of the first body, and the second body includes: a transfer hole formed at a position corresponding to the side chamber; and a connection part that connects the second body to be rotatable independently from the first body, and the other side of the lower reagent moving means can be fixed to the lower part of the transfer hole.

In one embodiment, the lower reagent transfer means is integrated with the second body and rotates, and by rotation of the second body, the chamber connected to the upper part of the transfer hole and the central chamber communicate through the lower reagent transfer means. It can be.

In one embodiment, a cartridge bottom packing rubber is included between the first body and the second body, and the cartridge bottom packing rubber is in close contact with the first body even when the second body rotates, so that the side chamber This may be to prevent the cargo inside from leaking out.

In one embodiment, the cartridge bottom packing rubber has an outlet hole formed at the center of the side chamber, and the side chamber may be connected to the lower reagent moving means through the outlet hole and the transfer hole.

In one embodiment, part or all of the central chamber may have a shape whose diameter narrows toward the bottom.

The present invention also includes a first body in which a plurality of side chambers are arranged outside the central chamber; a central chamber located in the center of the first body; a lower reagent moving means that rotates at the bottom of the central chamber to connect the central chamber and the side chamber; A gene extraction and detection method using a gene extraction and detection cartridge including a pump connected to the upper part of the central chamber to pressurize or depressurize the interior of the central chamber, (a) inserting a sample tube containing a sample into the cartridge connecting with one of the side chambers of; (b) Rotating the lower reagent moving means to connect one or more of the side chambers with the central chamber, then depressurizing the central chamber to transfer the sample, reagent, or magnetic bead in the side chamber to the central chamber for reaction. , after the reaction is completed, pressurizing the central chamber to discharge excess reagent into the side chamber; (c) repeating step (b) to extract genes; and (d) pressurizing the central chamber to transfer the extracted gene to a PCR channel.

In one embodiment, step (b) includes: (i) rotating the lower reagent moving means to connect it to the collection reagent chamber and depressurizing the central chamber to move the germ collecting reagent into the central chamber; (ii) rotating the lower reagent moving means to connect to the magnetic bead chamber and depressurizing the central chamber to transfer the magnetic beads to the central chamber; (iii) rotating the lower reagent moving means to connect the injection chamber connected to the sample tube and depressurizing the central chamber to move the sample in the sample tube to the central chamber; (iv) depressurizing and pressurizing the central chamber to mix the sample, the reagent, and the magnetic beads in the central chamber; (v) fixing the magnetic beads by contacting a magnet to the lower part of the cartridge and pressurizing the central chamber to discharge excess reagent; (vi) rotating the lower reagent moving means to connect to the cleaning chamber and depressurizing and pressurizing the central chamber to clean the magnetic beads inside the central chamber; and (vii) rotating the lower reagent moving means to connect to the nucleic acid elution chamber, decompressing the central chamber, and moving the nucleic acid elution buffer into the central chamber to extract genes.

In one embodiment, in steps (v) and (vi), the magnetic beads may be fixed to the inside of the lower reagent moving means by the magnetic material.

In one embodiment, step (vi) may be repeated 1 to 10 times.

The cartridge for gene extraction and detection according to the present invention is a sealed type using technology to directly isolate/concentrate sepsis-causing infectious agents from whole blood without a separate culture process and fluorescence detection of multiplex nested PCR amplification products of multiple genes. By implementing it in an automated manner in a cartridge, information on the causative bacteria and resistance genes of sepsis can be derived within 4 hours from whole blood, and the problem of antibiotic misuse caused by existing diagnostic methods can be improved.

In addition, the cartridge for gene extraction and detection according to the present invention sequentially performs the steps of separation, concentration, washing, elution, and PCR, so it provides a different type of analysis compared to existing analysis methods that proceed with the steps of elution, DNA collection, washing, and PCR. Decreased sensitivity due to DNA contained in cells (for example, human cells) can be minimized.

In addition, the cartridge for gene extraction and detection according to the present invention can automatically isolate bacteria and perform PCR through one cartridge, making it easy to isolate causative bacteria and select antibiotics according to resistance without manipulation by an expert. You can.

Figure 1 shows a cartridge for gene extraction and detection according to an embodiment of the present invention.

Figure 2 shows the connection of the sample tube to the cartridge for gene extraction and detection according to an embodiment of the present invention, where (a) shows the separated state and (b) shows the connected state, respectively.

Figure 3 shows the cap removed from the cartridge for gene extraction and detection according to an embodiment of the present invention.

Figure 4 shows a disassembled cartridge for gene extraction and detection according to an embodiment of the present invention.

Figure 5 shows the bottom surface of a cartridge for gene extraction and detection according to an embodiment of the present invention.

Figure 6 shows a cross section of a cartridge for gene extraction and detection according to an embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted. Throughout the specification, singular expressions should be understood to include plural expressions, unless the context clearly indicates otherwise, and terms such as “comprise” or “have” refer to the features, numbers, steps, operations, or components being described. , it is intended to specify the existence of a part or a combination thereof, but should be understood as not excluding in advance the possibility of the existence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof. . In addition, when performing a method or manufacturing method, each process forming the method may occur differently from the specified order unless a specific order is clearly stated in the context. That is, each process may occur in the same order as specified, may be performed substantially simultaneously, or may be performed in the opposite order.

The technology disclosed in this specification is not limited to the implementation examples described herein and may be embodied in other forms. However, the implementation examples introduced here are provided to ensure that the disclosed content is thorough and complete and that the technical idea of the present technology can be sufficiently conveyed to those skilled in the art. In order to clearly express the components of each device in the drawing, the sizes of the components, such as width and thickness, are shown somewhat enlarged. When describing the drawing as a whole, it is described from the observer's point of view, and when an element is mentioned as being located above another element, this means that the element may be located directly above another element or that additional elements may be interposed between those elements. Includes. Additionally, those skilled in the art will be able to implement the idea of the present invention in various other forms without departing from the technical idea of the present invention. In addition, the same symbols in a plurality of drawings refer to substantially the same elements.

As used herein, the term 'and/or' includes a combination of a plurality of listed items or any of a plurality of listed items. In this specification, 'A or B' may include 'A', 'B', or 'both A and B'.

Figure 1 shows a cartridge for gene extraction and detection of the present invention.

The present invention includes a first body in which a plurality of side chambers are arranged outside the central chamber; a central chamber located in the center of the first body; a lower reagent moving means that rotates at the bottom of the central chamber to connect the central chamber and the side chamber; and a pump connected to the upper part of the central chamber to pressurize or depressurize the interior of the central chamber.

The first body 100 may be manufactured in a shape in which a plurality of side chambers 110 are arranged outside the central chamber 310. The side chambers 110 provide convenience in manufacturing and assembly. For this purpose, it can be manufactured in a way that the lower part is connected to the first body 100.

That is, in the case of the side chambers 110, the lower parts are connected to form a donut shape, and the upper parts are molded to form each side chamber 110, so that the first body 100 can be manufactured as an integrated piece.

The side chamber 110 is a chamber for loading reagents and magnetic beads for gene extraction, which will be described later. A large number of chambers are arranged sequentially, so that each reagent can be used sequentially.

Looking at this in detail, the plurality of side chambers 110 are; An injection chamber 111 to which the sample tube is coupled; A collection reagent chamber 112 loaded with a solution for collecting bacteria; A magnetic bead chamber 113 loaded with a magnetic bead light buffer solution; A cleaning chamber 114 loaded with a cleaning solution; A nucleic acid extraction chamber 115 loaded with a buffer for nucleic acid elution; And it may include a connection chamber 116 to which a PCR channel 400 is connected (see FIG. 3).

The injection chamber 111 is a part where the sample tube loaded with the sample is coupled, and unlike the other side chambers 110 and the central chamber 310, which are sealed, the top is open so that the sample tube 500 can be coupled. there is. At this time, it is preferable that the top of the injection chamber 111 is cut diagonally so that it can be coupled to the sample tube 500 by penetrating through the top of the sample tube.

When a sample is inhaled through a single injection port, negative pressure is applied inside the sample container, making it difficult for the sample to come out of the container, and the desired amount of sample may not be obtained. Therefore, by forming an additional injection port on the side of the injection chamber 111, it is possible to absorb the sample as much as possible by relieving the negative pressure inside the sample.

The sample tube 500 can be used without limitation as long as it can collect and store a sample. Preferably, a tube-type or test-tube-type container whose inlet is sealed with a polymer membrane can be used (see FIG. 2).

The sample is collected from a patient suspected of being infected and may include blood, urine, tears, nasal discharge, saliva, sweat, exudate, tissue, or stool. In addition, samples may be collected not only directly from the patient as described above, but also from objects around the patient.

The collection reagent chamber 112 is a part where a solution for collecting bacteria is loaded. Due to the nature of the bacteria collection step that requires the largest amount of solution, the volume of the collection reagent chamber 112 may be the largest in the side chamber 110. There is (see Figure 3). However, in the case of the first body 100, it is easy to store and move because it is shaped like a donut with the central chamber 310 as the center. Therefore, in the case of the collection reagent chamber 112, it is formed along the outer peripheral surface of the central chamber 310. It can be formed to form an arc shape.

In addition, in the case of the side chamber 110, which will be described later, used reagents can be removed by reinjecting them into each chamber, but it is also possible to store used reagents by using the collection reagent chamber 112 as a disposal chamber. possible. This is because the internal volume of the collection reagent chamber 112 is manufactured to be larger than the total of the other side chambers 110, so that it can have a sufficient volume to store the reagent after use, and the solution for collecting bacteria is first moved to the central chamber 310. Therefore, it is easy to store used reagents. Additionally, the overall volume of the cartridge can be reduced by not allocating a separate disposal chamber for used reagents. When using the collection reagent chamber 112 as a disposal chamber as described above, it is desirable to make the internal volume of the collection reagent chamber 112 1.1 to 2 times the volume of the solution for collecting bacteria. Through this, it is possible to load not only the used solution for collecting bacteria but also other used reagents.

The magnetic bead chamber 113 is a chamber in which magnetic beads and a buffer solution are loaded. The magnetic bead may be a bead-shaped particle with a magnetic core inside, and a magnetic bead coated with apolipoprotein H (ApoH) on the shell portion may be used. In the case of the apolipoprotein, ApoH can bind with high affinity to lipopolysaccharide (LPS), a major component of the outer wall of Gram-negative bacteria, and to specific proteins of Gram-positive pathogens, so when using magnetic beads as above. It is possible to selectively isolate Gram-positive or Gram-negative bacteria and fungi from whole blood.

The buffer solution is used to store and transport the magnetic beads, and serves to prevent damage when storing the magnetic beads in the form of particles and to facilitate movement when moving by depressurizing the central chamber 310, which will be described later. can do. The buffer solution can be used without limitation as long as it satisfies these conditions, but water or physiological saline can be preferably used.

The cleaning chamber 114 is a chamber in which a cleaning solution is loaded. One cleaning chamber 114 can be used, but for smooth cleaning, it can be divided into 1 to 10, preferably 2 to 5, cleaning chambers 114. It can be configured.

The cleaning solution used for the above cleaning can be used without limitation as long as it can remove excess blood or human secretions. Preferably, water, saline solution, PBS, Tris-EDTA, etc. can be used.

In addition, when it is composed of 1 to 10 washing chambers 114 as described above, washing can be repeated 1 to 10 times as will be described later, and through this, cleaning of other foreign substances other than bacteria can be performed smoothly. . However, when more than 10 cleaning chambers 114 are used, it is uneconomical because a lot of time is required for cleaning and the size of the cartridge becomes excessively large.

The nucleic acid extraction chamber 115 is a chamber loaded with a buffer for nucleic acid extraction. When cleaning is completed as described above, magnetic beads combined with causative bacteria may remain in the central chamber 310. In the case of the present invention, since the type of the causative bacteria and antibiotic resistance are confirmed by analyzing the RNA or DNA present in the causative bacteria, it is preferable to hemolyze the causative bacteria and extract hexane. Therefore, after the washing is completed as described above, the nucleic acid extraction buffer can be supplied to hemolyze the causative bacteria and simultaneously extract the nucleic acid. At this time, the nucleic acid extraction buffer can be used without limitation as long as it is a buffer capable of hemolyzing the causative bacteria.

The connection chamber 116, like the central chamber 310, is installed to connect the PCR channel 400 and is a chamber that moves the extracted nucleic acid as described above to the PCR channel 400. Unlike the other side chambers 110, the connection chamber 116 does not load specific chemicals and is used as a simple movement passage, so it is preferable that the inner diameter is smaller than that of the other side chambers 110.

The PCR channel 400 is a channel for performing PCR. Unlike existing PCR devices, the PCR channel 400 is manufactured in the form of a lab-on-a-chip and may include a plurality of channels therein. That is, the nucleic acid supplied to the PCR channel 400 can enter the inside of the PCR channel and undergo PCR, and thus the nucleic acid can be amplified and analyzed at the same time. In particular, in the case of the present invention, the antibiotic resistance of the causative bacteria can be confirmed by providing a means to detect antibiotic resistance genes, and since nucleic acids appear differently depending on each causative bacteria, it is possible to select an antibiotic optimized for the causative bacteria (Figure 1 and Figure 3).

In addition, in the case of the PCR channel 400, it is possible to manufacture it in a form fixed to the connection chamber 116, but it is preferably manufactured in a separate form so that the appropriate PCR channel 400 can be selected and used according to each causative bacteria. It is preferable, and in particular, one side of the PCR channel 400 is connected to one side of the lid, and one lower end of the PCR channel 400 can be manufactured in a format that is secured to the connection chamber 116. Through this, the PCR channel 400 can be stored separately from the cartridge and can be combined with the cartridge to facilitate storage of the cartridge. In addition, the PCR channel 400 may be manufactured as an integrated or combined type as above, but it is also possible to manufacture the PCR channel 400 and the connection chamber 116 with a pipe. In this case, the PCR channel 400 can be installed and operated independently of the cartridge.

As seen above, the first body 100 may be composed of a plurality of side chambers 110. However, for convenience in manufacturing, since the first body 100 is manufactured with the top open, it is preferable to use a lid 200 that can close the top of the side chamber 110.

The lid 200 may be coupled to the upper part of the first body 100 to close the upper part of the central chamber 310 and the side chamber 110 (see FIGS. 3 and 4). The lid 200 can be largely divided into two parts. One is a part used to close the central chamber 310 (inner lid 220), and the other is a part used to close the side chamber 110 (outer lid 210). These two parts can be fitted into one piece, but it is preferable that they are coupled so that they can rotate independently.

Looking at this in detail, as will be described later, the central chamber 310 can be integrated with the second body 300 and rotate. The second body 300 is located below the first body 100 and rotates integrally with the lower reagent moving means 320, and is consequently used to close the top of the central chamber 310. The portion also rotates together with the lower reagent moving means 320. At this time, when marking 230 is made on the part used to close the upper part of the central chamber 310 and a certain notch is engraved on the part used to close the side chamber 110, the position of the lower reagent moving means 320 is determined. It is possible to easily observe from the top (see Figure 3).

Since the position of the lower reagent moving means 320 can indicate the reagent currently being used, it is possible to easily visually check the current progress with such a simple indicator.

Additionally, a pump connection portion 240 to which the pump is connected may be formed in the center of the lid. The pump is a part that moves fluid by depressurizing or pressurizing the inside of the central chamber 310. Therefore, the pump can be connected to the central chamber 310 through the pump connection part 240 formed on the lid, and for this purpose, the pump connection part 240 is formed at the center of the lid, especially the center of the inner lid 220. It can be.

Looking at this in detail, the inner lid 220 can be combined with the central chamber 310 to seal the upper part of the central chamber 310. At this time, since the central chamber 310 is connected to the side chamber 110 connected through the lower reagent moving means 320, when the central chamber 310 is depressurized using the pump, the side chamber 110 The reagent loaded in may be moved to the central chamber 310. On the contrary, when the inside of the central chamber 310 is pressurized, it is possible to move the reagent inside the central chamber 310 toward the side chamber 110. This pressurization or decompression may be used to move the reagent back and forth, but it is also possible to mix the reagents by repeating the pressurization or decompression.

The pump can be used without limitation as long as it can depressurize or pressurize the inside of the central chamber 310, but a syringe pump, a reciprocating pump, a rotary pump, etc. may be used, and a syringe pump may be preferably used. In particular, in the case of the present invention, since it is necessary to prevent foreign substances from entering the interior of the central chamber 310, it is desirable to use a syringe pump that can pressurize and depressurize using only internal air.

The lower reagent moving means 320 is a part that selectively connects the lower part of the side chamber 110 and the central chamber 310 and serves as a passage for reagents.

Looking at this in detail, a moving channel may be formed inside the lower reagent moving means 320, and one side of the channel may be fixed to the lower center of the central chamber 310. Additionally, the other side of the moving channel may be connected to one or more of the plurality of side chambers 110. At this time, in the case of the lower reagent moving means 320, as seen above, it is integrated with the second body 300 and can rotate, so it rotates so that the desired side chamber 110 is located at the upper end of the other side. 310) can be connected to the desired side chamber 110 (see FIG. 5). In addition, in the case of the central chamber 310, since it is integrated with the second body 300 and rotates, the connection can be maintained regardless of whether the lower reagent moving means 320 rotates, but preferably, the movement channel One side may be connected to the center of the central chamber 310. Through this, the reagent supplied from the side chamber 110 can be evenly supplied to the central chamber 310, and through this, a certain reaction can be performed.

In addition, the position of the lower reagent moving means 320 can be easily observed from the top by the marking on the inner lid 220, as shown above, so the completion or progress of the reaction can be easily observed using this. You can.

Additionally, the lower reagent moving means 320 may include a moving channel therein. In the case of the moving channel, it can be a passage through which the reagent moves. In addition, by having a mixing structure inside the channel, movement and mixing of the reagent can be performed simultaneously, and if a valve is installed in some of the channels, the amount of movement can be limited. Separately, when loading additional reagents into some channels, it is possible to mix additional chemicals with the reagents and supply them.

A second body 300 is coupled to the lower part of the first body 100, and the second body 300 includes a transfer hole 321 formed at a position corresponding to the side chamber 110; And a connection part that connects the second body 300 to be rotatable independently from the first body 100, and the other side of the lower reagent moving means 320 can be fixed to the lower part of the transfer hole. .

The second body 300 is coupled to the lower part of the first body 100 and is a part that connects the first body 100 and the lower reagent moving means 320. At this time, the central chamber 310 may be connected to the center of the second body 300. As seen above, the central chamber 310 is fixed to the second body 300, so the second body 300 It can be rotated together by the rotation of .

A transfer hole 321 may be formed in the second body 300. The side chambers 110 are arranged along the outer peripheral surface of the central chamber 310, and in this case, may be arranged to have the same distance from the center of the central chamber 310. Therefore, when the transfer hole 321 is formed at a position corresponding to the side chamber 110, the transfer hole 321 is located at the lower part of each side chamber 110 as the second body 300 rotates. It is possible to do so, and thus the lower part of each side chamber 110 can communicate with the lower reagent moving means 320 (see FIG. 5).

In addition, the other side of the lower reagent moving means 320 may be connected to the lower part of the transfer hole 321. That is, the lower reagent transfer means is integrated with the second body 300 and rotates, and the rotation of the second body 300 causes the chamber connected to the upper part of the transfer hole and the central chamber 310 to move the lower reagent. It can be communicated through means 320.

The connection part allows the second body 300 to be coupled to the lower part of the first body 100, and the second body 300 can be rotatably coupled. At this time, the connection can be used without limitation as long as it has a structure capable of fixing the second body 300 as above, but preferably, a rail is installed along the lower outer peripheral surface of the first body 100, and the second body ( A latch may be formed on the protrusion extending to the top of 300 so that it can be fitted with the rail of the first body 100. Through this, it is possible to couple the protrusion of the second body 300 to rotate along the rail of the first body 100 (see FIGS. 3 and 4).

It includes a cartridge bottom packing rubber 620 between the first body 100 and the second body 300, and the cartridge bottom packing rubber 620 remains in contact with the second body 300 even when the second body 300 rotates. It may be in close contact with the first body 100 to prevent the load inside the side chamber 110 from leaking out (see FIG. 4).

As seen above, the second body 300 may be rotatably connected to the lower part of the first body 100. However, when the second body 300 is rotatably connected, sealing of the side chamber 110 installed in the first body 100 may be problematic. In addition, even if sealing is possible, reagents may leak out due to rotation of the second body 300, and even if only a slight error occurs during rotation, smooth transfer of reagents may not be achieved. Therefore, a bottom packing rubber 620 is added between the first body 100 and the second body 300 to seal the lower part of the side chamber 110 and at the same time smoothly connect the chamber and the transfer hole. You can do it.

The lower packing rubber 620 is coupled to the lower part of the first body 100 and is fixed to the first body 100 so that it remains attached to the first body 100 even when the second body 300 rotates. It can be tightly fixed. Through this, the lower end of the side chamber 110 of the first body 100 can be sealed, and an outlet hole can be formed in the center of each side chamber 110. That is, the side chamber 110 may be connected to the lower reagent moving means 320 through the outlet hole and the transfer hole.

Additionally, a top packing rubber 610 can be installed between the first body 100 and the lid to prevent reagents from leaking between the first body 100 and the lid (see FIG. 4).

In the case of the central chamber 310, part or all of it may have a shape whose diameter narrows toward the bottom (see FIG. 6). As seen above, a supply hole 322 connected to the lower reagent moving means 320 may be formed in the lower central portion of the central chamber 310. However, in the case of this supply hole 322, not only does it serve to supply the reagent in the direction of the central chamber 310, but it can also transport the reaction completed reagent in the central chamber 310 to the outside. Therefore, in order to facilitate this transfer, it is desirable that part or all of the central chamber 310 be manufactured in a shape where the diameter narrows toward the bottom. That is, the lower part of the central chamber 310 is manufactured in a funnel shape so that the fluid inside can be smoothly discharged in the direction of the supply hole 322.

Hereinafter, the present invention will be described in detail through gene extraction and detection methods.

The present invention also includes a first body 100 in which a plurality of side chambers 110 are arranged outside the central chamber 310; A central chamber 310 located in the center of the first body 100; a lower reagent moving means 320 that rotates at the bottom of the central chamber 310 and connects the central chamber 310 and the side chambers 110; A gene extraction and detection method using a gene extraction and detection cartridge including a pump connected to the upper part of the central chamber 310 to pressurize or depressurize the interior of the central chamber 310, including (a) a sample. connecting a sample tube to one of the side chambers 110 of the cartridge; (b) connect one or more of the side chambers 110 with the central chamber 310 by rotating the lower reagent moving means, and then depressurize the central chamber 310 to obtain a sample in the side chamber 110, transferring reagents or magnetic beads to the central chamber 310 to react, and after the reaction is completed, pressurizing the central chamber 310 to discharge excess reagents into the side chamber 110; (c) repeating step (b) to extract genes; and (d) transferring the extracted gene to the PCR channel 400 by pressurizing the central chamber 310.

The step (a) is a step of connecting a sample tube containing a sample to one of the side chambers 110 of the cartridge, collecting a sample, then injecting it into the sample tube, and inserting the sample tube containing the sample into the side chamber ( This is the step of connecting to the injection chamber 111, which is one of 110). At this time, since one side of the sample tube is sealed with a polymer resin membrane, it can be inserted from the top to the bottom of the injection chamber 111 with the polymer membrane at the bottom.

In step (b), the samples and reagents loaded in each side chamber 110 are moved to the central chamber 310 to react. In step (b), one or more of the side chambers 110 are connected to the central chamber 310 by rotating the lower reagent moving means, and then the central chamber 310 is depressurized to form the side chamber 110. It may include transferring the sample, reagent, or magnetic bead within the central chamber 310 to react, and after the reaction is completed, pressurizing the central chamber 310 to discharge excess reagent into the side chamber 110, As in step (c) above, this can be repeated several times to sequentially perform steps such as reaction, washing, and hemolysis.

Looking at step (b) in detail, step (b) includes (i) rotating the lower reagent moving means to connect it to the collection reagent chamber 112 and depressurizing the central chamber 310 to collect a reagent for collecting bacteria. moving to the central chamber 310; (ii) rotating the lower reagent moving means to connect to the magnetic bead chamber 113 and depressurizing the central chamber 310 to transfer the magnetic beads to the central chamber 310; (iii) rotating the lower reagent moving means to connect the injection chamber 111 connected to the sample tube and depressurizing the central chamber 310 to move the sample in the sample tube to the central chamber 310; (iv) depressurizing and pressurizing the central chamber 310 to mix the sample, the reagent, and the magnetic beads in the central chamber 310; (v) fixing the magnetic beads by contacting a magnet to the lower part of the cartridge and then pressurizing the central chamber 310 to discharge excess reagent into the collection reagent chamber 112; (vi) rotating the lower reagent moving means to connect to the cleaning chamber 114 and depressurizing and pressurizing the central chamber 310 to clean the magnetic beads inside the central chamber 310; and (vii) rotating the lower reagent moving means to connect to the nucleic acid elution chamber 115 and decompressing the central chamber 310 to move the nucleic acid elution buffer to the central chamber 310 to extract genes. It can be included.

Step (i) is a step in which the lower reagent moving means is rotated to connect to the collection reagent chamber 112 and the central chamber 310 is depressurized to move the germ-collecting reagent to the central chamber 310. In the case of the central chamber 310, it may be initially supplied without any reagents loaded therein. Therefore, by supplying the collection reagent first as described above, it is possible to prevent the magnetic beads and samples to be supplied later from adhering to the surface of the central chamber 310. At this time, the movement of the collection reagent can be performed by depressurizing the inside of the central chamber 310 using a pump connected to the upper part of the central chamber 310, as described above, and the central chamber (310) as described above. 310) When the internal pressure is reduced, the collection reagent can be moved from the collection reagent chamber 112 to the central chamber 310 through the lower reagent moving means 320.

Step (ii) is a step of rotating the lower reagent moving means to connect to the magnetic bead chamber 113 and depressurizing the central chamber 310 to transfer the magnetic beads to the central chamber 310.

After the collection reagent is supplied to the central chamber 310 as described above, the second body 300 is rotated so that the other side of the lower reagent moving means 320 is positioned at the bottom of the magnetic bead chamber 113. It can be connected to (113). Thereafter, in the same manner as step (i), the central chamber 310 may be depressurized to transfer the magnetic beads in the magnetic bead chamber 113 to the central chamber 310.

However, since the magnetic beads have a particle shape, they can aggregate and sink to the bottom of the magnetic bead chamber 113, so the magnetic beads can be agglomerated by pressurizing and depressurizing the central chamber 310. can be resolved.

Looking at this in detail, after the other side of the lower reagent moving means 320 is connected to the magnetic bead chamber 113, a portion of the collection reagent can be supplied to the magnetic bead chamber 113 by pressurizing the central chamber 310. there is. At this time, since the collection reagent is supplied to the magnetic bead chamber 113 at a constant flow rate, agglomeration of the magnetic beads can be resolved by the flow rate of the collection reagent. Afterwards, when the pressure inside the central chamber 310 is reduced, even the collection reagent mixed with the magnetic beads can be moved to the central chamber 310. In addition, if the aggregation of the magnetic beads is severe or the aggregation is not resolved by supplying the collection reagent, it is preferable to repeat the pressurization and decompression of the central chamber 310 2 to 5 times. Through this, the collection reagent can be repeatedly supplied and sucked into the magnetic bead chamber 113 2 to 5 times, and agglomeration of the magnetic beads can be resolved by the flow of the collection reagent.

Step (iii) includes rotating the lower reagent moving means to connect the injection chamber 111 connected to the sample tube and depressurizing the central chamber 310 to move the sample in the sample tube to the central chamber 310. am.

When step (ii) is completed, the collection reagent and the magnetic beads may be mixed and present inside the central chamber 310. Afterwards, when the sample in the sample tube is supplied to the central chamber 310 as described above, the causative bacteria in the sample may attach to the surface of the magnetic beads due to the collection reagent. In addition, as seen above, since the surface of the magnetic bead is coated with ApoH, it is possible for Gram-positive bacteria or Gram-negative bacteria and fungi to selectively attach to the surface of the magnetic bead.

Step (iv) is a step of mixing the sample, the reagent, and the magnetic beads in the central chamber 310 by depressurizing and pressurizing the central chamber 310.

As described above, the interior of the central chamber 310 is preferably agitated to facilitate attachment of the causative bacteria to the magnetic beads, and this may be performed by pressurizing or depressurizing the interior of the chamber. Looking at this in detail, after the sample is supplied as described above, the second body 300 can be rotated to connect the other side of the lower reagent moving means 320 to the collection reagent chamber 112. As seen above, the collection reagent chamber 112 is manufactured larger than the other side chambers 110, so it is preferable to mix using the collection reagent chamber 112. After the collection reagent chamber 112 is connected, the central chamber 310 can be pressed to move some or all of the samples, reagents, and magnetic beads in the central chamber 310 to the collection reagent chamber 112, By depressurizing the central chamber 310, the sample, reagent, and magnetic beads in the collection reagent chamber 112 can be moved to the central chamber 310. Through this movement, mixing of the sample, reagent, and magnetic beads can be performed uniformly. In addition, it is possible to repeat the above movement several times to make this mixing more uniform and to increase the probability that causative bacteria such as bacteria and fungi will encounter ApoH-coated magnetic beads.

In addition, the mixing of the sample, the reagent, and the magnetic beads as described above is preferably performed over 15 to 60 minutes, and may be performed at 30 to 50°C, preferably 33 to 40°C. If it is less than the above range, the reaction may not be completed and the number of causative bacteria captured in the magnetic particles may decrease, and if it exceeds the above range, the protein may be denatured and the amount of adhesion may decrease.

Step (v) is a step of fixing the magnetic beads by contacting a magnet to the bottom of the cartridge and pressurizing the central chamber 310 to discharge excess reagent.

When stirring is completed as described above, causative bacteria present in the sample may attach to the surface of the magnetic beads. At this time, as shown above, if the central chamber 310 is pressurized to discharge the reagent whose reaction has been completed, the magnetic beads can also be discharged. Therefore, a magnet can be brought into contact with the lower part of the cartridge to fix the magnetic bead.

That is, in steps (v) and (vi), the magnetic beads may be fixed to the inside of the lower reagent moving means by the magnetic material. When the central chamber 310 is pressurized, most of the magnetic beads are fixed by the magnet while passing through the lower reagent moving means 320, and only the reagent that has completed the reaction can be discharged to the outside.

In addition, it is preferable that the used reagents are discharged in the direction of the collection reagent chamber 112.

Step (vi) is a step of rotating the lower reagent moving means to connect it to the cleaning chamber 114 and depressurizing and pressurizing the central chamber 310 to clean the magnetic beads inside the lower reagent moving means 320.

Even after the reaction completion reagent is discharged as described above, a part of the unnecessary reaction completion reagent and a part of the sample may remain in the lower reagent moving means 320, in addition to the magnetic beads to which the causative bacteria are attached. Therefore, by washing and removing it, the efficiency of PCR to be performed later can be increased. To this end, the second body 300 is rotated to connect the lower reagent moving means 320 to the cleaning chamber 114, and then the cleaning solution in the cleaning chamber 114 is moved to the central chamber 310. You can do it. Also, in this case, the cleaning efficiency can be increased by repeating the movement of the upper cleaning liquid 2 to 5 times as in the mixing step, and the cleaning liquid used in the final discharge step can be discharged to the cleaning chamber 114 or the collection chamber ( 112). After this step, the collection chamber 112 can be operated as a waste chamber.

In addition, the above washing may be repeated 2 to 10 times. To this end, the number of cleaning chambers 114 may be 2 to 10, preferably 2 to 5.

In step (vii), the lower reagent moving means is rotated to connect to the nucleic acid elution chamber, the central chamber 310 is decompressed, and the nucleic acid elution buffer is moved to the central chamber 310 to extract genes.

After cleaning is completed as described above, only the causative bacteria attached to the magnetic particles may remain inside the lower reagent transfer means 320. Therefore, by supplying a buffer for nucleic acid elution as described above, the causative bacteria can be hemolyzed and nucleic acids can be leaked to the outside. In addition, the buffer for nucleic acid elution may act as a solution for moving nucleic acids in a step to be described later, and therefore, unlike the above collection solution or washing solution, it may not be moved to a disposal chamber (collection chamber).

Additionally, the elution of nucleic acids as described above may be performed at a temperature of 60 to 120°C, preferably 85 to 100°C for 1 to 15 minutes. Below the above range, the amount of nucleic acid eluted may be reduced due to insufficient hemolysis of the causative bacteria, and if it exceeds the above range, the eluted nucleic acid may be thermally deformed, resulting in increased false negatives.

When step (b) is completed as described above, RNA or DNA mixed with a buffer for nucleic acid elution may exist inside the central chamber 310. At this time, PCR can be performed by pressurizing the central chamber 310 and transferring the extracted gene to the PCR channel 400 (step (d)). At this time, the second body 300 can be rotated to connect the lower reagent moving means 320 to the connection chamber 116, and magnetic particles still remain in the lower reagent moving means 320. It is preferable to attach a magnet to the lower part of the cartridge to fix the magnetic particles inside the lower reagent moving means 320.

In addition, in the case of the PCR chamber, the sensitivity may decrease if foreign substances other than RNA or DNA are introduced, and a filter through which RNA and DNA can pass is installed in the connection chamber 116 to prevent the inflow of magnetic particles and unhemolyzed cells. It is desirable to block it.

When the nucleic acids (RNA and DNA) are supplied to the PCR chamber as described above, PCR can be performed, and each gene can be analyzed to confirm the type of causative bacteria and the presence or absence of antibiotic resistance genes.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (15)

a first body in which a plurality of side chambers are arranged outside the central chamber; a central chamber located in the center of the first body; a lower reagent moving means that rotates at the bottom of the central chamber to connect the central chamber and the side chamber; and a pump connected to the upper part of the central chamber to pressurize or depressurize the interior of the central chamber; A cartridge for gene extraction and detection comprising. According to paragraph 1, The plurality of side chambers; an injection chamber into which the sample tube is coupled; A collection reagent chamber loaded with a solution for collecting bacteria; A magnetic bead chamber loaded with a magnetic bead light buffer solution; A cleaning chamber loaded with a cleaning solution; A nucleic acid extraction chamber loaded with a buffer for nucleic acid elution; and A connection chamber with a PCR channel connected to the top; A cartridge for gene extraction and detection comprising. According to paragraph 1, A lid that closes the upper part of the central chamber and the side chamber is coupled to the upper part of the first body, A cartridge for gene extraction and detection in which a pump connection part to which the pump is connected is formed in the center of the lid. According to paragraph 3, One side of the PCR channel is connected to one side of the lid, A cartridge for gene extraction and detection is connected to a connection chamber at the bottom of one side of the PCR channel. According to paragraph 1, The lower reagent moving means includes a moving channel therein, A cartridge for gene extraction and detection in which one side of the moving channel is fixed to the center of the central chamber, and the other side is selectively connected to the plurality of side chambers according to rotation of the lower reagent moving means. According to clause 5, The lower reagent moving means is, A cartridge for gene extraction and detection that moves samples, reagents, and magnetic beads between the central chamber and the side chamber through the pressure that the central chamber is pressurized or depressurized. According to paragraph 1, A second body is coupled to the lower part of the first body, The second body is, a transfer hole formed at a position corresponding to the side chamber; and a connection portion that connects the second body to be rotatable independently from the first body; Includes, A cartridge for gene extraction and detection in which the other side of the lower reagent moving means is fixed to the lower part of the transfer hole. In clause 7, The lower reagent transport means is integrated with the second body and rotates, A cartridge for gene extraction and detection in which a chamber connected to the upper part of the transfer hole and the central chamber communicate through the lower reagent moving means by rotation of the second body. In clause 7, Includes a cartridge bottom packing rubber between the first body and the second body, The cartridge bottom packing rubber is in close contact with the first body even when the second body rotates to prevent the load inside the side chamber from leaking. According to clause 9, The cartridge bottom packing rubber is, An outlet hole is formed at the center of the side chamber, A cartridge for gene extraction and detection wherein the side chamber is connected to the lower reagent moving means through the outflow hole and the transfer hole. According to paragraph 1, A cartridge for gene extraction and detection, wherein part or all of the central chamber has a shape whose diameter narrows toward the bottom. a first body in which a plurality of side chambers are arranged outside the central chamber; a central chamber located in the center of the first body; a lower reagent moving means that rotates at the bottom of the central chamber to connect the central chamber and the side chamber; A gene extraction and detection method using a gene extraction and detection cartridge including a pump connected to the upper part of the central chamber to pressurize or depressurize the interior of the central chamber, (a) connecting a sample tube containing a sample with one of the side chambers of the cartridge; (b) Rotating the lower reagent moving means to connect one or more of the side chambers with the central chamber, then depressurizing the central chamber to transfer the sample, reagent, or magnetic bead in the side chamber to the central chamber for reaction. , after the reaction is completed, pressurizing the central chamber to discharge excess reagent into the side chamber; (c) repeating step (b) to extract genes; and (d) transferring the extracted gene to the PCR channel by pressurizing the central chamber; Gene extraction and detection method comprising. According to clause 12, In step (b), (i) rotating the lower reagent moving means to connect it to the collection reagent chamber and depressurizing the central chamber to move the germ-collecting reagent into the central chamber; (ii) rotating the lower reagent moving means to connect the magnetic bead chamber and depressurizing the central chamber to transfer the magnetic beads to the central chamber; (iii) rotating the lower reagent moving means to connect the injection chamber connected to the sample tube and depressurizing the central chamber to move the sample in the sample tube to the central chamber; (iv) depressurizing and pressurizing the central chamber to mix the sample, the reagent, and the magnetic beads in the central chamber; (v) fixing the magnetic beads by contacting a magnetic material to the lower part of the cartridge and pressurizing the central chamber to discharge excess reagent; (vi) rotating the lower reagent moving means to connect it to a cleaning chamber and depressurizing and pressurizing the central chamber to clean the magnetic beads; and (vii) rotating the lower reagent moving means to connect to the nucleic acid elution chamber and decompressing the central chamber to move the nucleic acid elution buffer into the central chamber to extract genes; Gene extraction and detection method comprising. According to clause 13, In steps (v) and (vi), the magnetic beads are fixed to the inside of the lower reagent transfer means by the magnetic material. According to clause 13, Step (vi) is a gene extraction and detection method that is repeated 2 to 10 times.

Images