WO2024117299A1 - Système d'extraction et de détection d'un gène - Google Patents

Système d'extraction et de détection d'un gène Download PDF

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Publication number
WO2024117299A1
WO2024117299A1 PCT/KR2022/019180 KR2022019180W WO2024117299A1 WO 2024117299 A1 WO2024117299 A1 WO 2024117299A1 KR 2022019180 W KR2022019180 W KR 2022019180W WO 2024117299 A1 WO2024117299 A1 WO 2024117299A1
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chamber
central chamber
cartridge
reagent
moving means
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PCT/KR2022/019180
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English (en)
Korean (ko)
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나훈종
한정덕
이상윤
이준민
장지성
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주식회사 퀀타매트릭스
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Publication of WO2024117299A1 publication Critical patent/WO2024117299A1/fr

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  • the present invention relates to a system for gene extraction and detection, and more specifically, gene extraction and detection that enables early selection of antibiotics appropriate for a patient by identifying the bacteria or strain causing an infection and detecting multiple antibiotic resistance gene information. It's about the dragon system.
  • 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.
  • 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.
  • LPS lipopolysaccharide
  • 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. If magnetic beads are mixed with blood and reacted, if there is an infectious agent (sepsis-causing bacteria) in the blood, only the infectious agent can attach to the magnetic beads. If the magnetic beads are separated by a magnetic material, the infectious agent in the blood can be separated and concentrated.
  • infectious agent sepsis-causing bacteria
  • Nested polymerase chain reaction 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.
  • 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.
  • 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.
  • nested PCR can be used to increase the accuracy and reliability of multiple gene amplification of multiple pathogens that can cause sepsis.
  • 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. .
  • the present invention includes a cartridge in which a plurality of side chambers are arranged outside the central chamber and includes a PCR channel on one upper side; A first temperature control unit that maintains the space where the cartridge is installed at a constant temperature; a pump connected to the upper part of the cartridge to pressurize and depressurize the central chamber; A fixing part that secures the upper part of the cartridge; a rotating part that rotates the lower part of the cartridge to communicate with the side chamber and the central chamber at a desired position; A magnetic material that secures the magnetic beads inside the cartridge; a second temperature control unit that controls the temperature of the central chamber; A third temperature control unit that controls the temperature of the PCR channel; and a detection unit that detects fluorescence in the PCR channel after PCR is completed.
  • the cartridge 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 it is installed at the upper part of the central chamber and may include a pump connection part to which a pump is connected.
  • 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.
  • one lower end of the PCR channel may be in communication with a connection chamber.
  • a lid that closes the upper part of the central chamber and the side chamber is coupled to the upper part of the cartridge, and a pump connection portion to which the pump is connected may be formed in the center of the lid.
  • 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 moves along the plurality of sides as the lower reagent moving means rotates. Can be selectively connected to the chamber.
  • 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.
  • 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 may be fixed to the lower part of the transfer hole.
  • 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.
  • the gene extraction and detection system includes a blocking plate that spatially separates the upper and lower parts, and the first temperature control unit, the fixing unit, the third temperature control unit, and the detection unit are the blocking plates. It is located at the upper part of the plate, and the pump, the rotating part, and the magnetic body may be located at the lower part of the blocking plate.
  • the present invention also provides a cartridge in which a plurality of side chambers are arranged outside the central chamber and includes a PCR channel on one upper side; A first temperature control unit that maintains the space where the cartridge is installed at a constant temperature; a pump connected to the upper part of the cartridge to pressurize and depressurize the central chamber; A fixing part that secures the upper part of the cartridge; a rotating part that rotates the lower part of the cartridge to communicate with the side chamber and the central chamber at a desired position; A magnetic material that secures the magnetic beads inside the cartridge; a second temperature control unit that controls the internal temperature of the lower reagent moving means; A third temperature control unit that controls the temperature of the PCR channel; And a gene extraction and detection method using a genetic difference extraction and detection system including a detection unit that detects the fluorescence of the PCR channel after PCR is completed, (a) connecting a sample tube containing a sample with one of the side chambers of the cartridge.
  • step (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; (d) transferring the extracted gene to the PCR channel by pressurizing the central chamber; and (e) detecting the gene using fluorescence generated in a PCR channel.
  • 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 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
  • the magnetic beads may be fixed to the inside of the lower reagent moving means by the magnetic material.
  • step (vi) may be repeated 2 to 10 times.
  • the gene extraction and detection system according to the present invention is implemented in an automated manner in a sealed cartridge using technology to directly isolate/concentrate sepsis-causing infectious agents from whole blood and fluorescence detection of multiplex nested PCR amplification products of multiple genes. It is possible to derive information on the causative bacteria and resistance genes of sepsis within 4 hours, and can improve the problem of antibiotic misuse and abuse caused by existing diagnostic methods.
  • the gene extraction and detection system sequentially performs the steps of separation, concentration, washing, elution, and PCR, so it can be used to identify different types of cells compared to existing analysis methods that proceed with the steps of elution, DNA collection, washing, and PCR. Decreased sensitivity due to DNA contained in (for example, human cells) can be minimized.
  • the gene extraction and detection system 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. there is.
  • Figure 1 shows the configuration of a system for gene extraction and detection according to an embodiment of the present invention.
  • Figure 2 shows the guide duct removed from the system for gene extraction and detection according to an embodiment of the present invention.
  • Figure 3 shows a first temperature control unit according to an embodiment of the present invention.
  • Figure 4 shows the combination of a fixing part and a cartridge according to an embodiment of the present invention.
  • Figure 5 shows a rotating part according to an embodiment of the present invention.
  • Figure 6 shows a third temperature control unit according to an embodiment of the present invention.
  • Figure 7 shows a detection unit according to an embodiment of the present invention.
  • Figure 8 shows a cartridge for gene extraction and detection according to an embodiment of the present invention.
  • Figure 9 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 10 shows the cap removed from the cartridge for gene extraction and detection according to an embodiment of the present invention.
  • Figure 11 shows a disassembled cartridge for gene extraction and detection according to an embodiment of the present invention.
  • Figure 12 shows the bottom surface of a cartridge for gene extraction and detection according to an embodiment of the present invention.
  • Figure 13 shows a cross section of a cartridge for gene extraction and detection according to an embodiment of the present invention.
  • 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.
  • 'and/or' includes a combination of a plurality of listed items or any of a plurality of listed items.
  • 'A or B' may include 'A', 'B', or 'both A and B'.
  • Figure 1 shows the overall configuration of the gene extraction and detection system of the present invention
  • Figure 2 is a diagram showing the cartridge portion exposed by removing the induction duct of the first temperature control unit.
  • the present invention includes a cartridge in which a plurality of side chambers are arranged outside the central chamber and includes a PCR channel on one upper side; A first temperature control unit that maintains the space where the cartridge is installed at a constant temperature; a pump connected to the upper part of the cartridge to pressurize and depressurize the central chamber; A fixing part that secures the upper part of the cartridge; a rotating part that rotates the lower part of the cartridge to communicate with the side chamber and the central chamber at a desired position; A magnetic material that secures the magnetic beads inside the cartridge; a second temperature control unit that controls the temperature of the central chamber; A third temperature control unit that controls the temperature of the PCR channel; and a detection unit that detects fluorescence in the PCR channel after PCR is completed (see FIG. 1).
  • the cartridge 10 is a space where gene extraction and amplification by PCR of the present invention are performed, and a plurality of side chambers are arranged outside the central chamber, and may include a PCR channel on one upper side.
  • the cartridge 10 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 it is installed at the upper part of the central chamber and may include a pump connection part to which a pump is connected.
  • 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.
  • 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.
  • 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. 10).
  • 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, the top of the injection chamber 111 is preferably cut diagonally so that it can be coupled to the sample tube 500 by penetrating through the top of the sample tube.
  • the sample tube 500 can be used without limitation as long as it can collect and store a sample.
  • a tube-type or test-tube-type container whose inlet is sealed with a polymer membrane can be used (see FIG. 9).
  • 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 10). 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.
  • 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.
  • 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.
  • ApoH apolipoprotein H
  • 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. Although one cleaning chamber 114 can be used, for smooth cleaning, it consists of 1 to 10 cleaning chambers 114, preferably 2 to 5 cleaning chambers 114. It can be.
  • the cleaning solution used for the above cleaning can be used without limitation as long as it can remove excess blood or human secretions.
  • water, saline solution, PBS, Tris-EDTA, etc. can be used.
  • 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.
  • more than 10 cleaning chambers 114 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.
  • 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.
  • the nucleic acid extraction buffer can be supplied to hemolyze the causative bacteria and simultaneously extract the nucleic acid.
  • the nucleic acid extraction buffer can be used without limitation as long as it is a buffer capable of hemolyzing the causative bacteria.
  • 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.
  • 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 8 and Figure 10).
  • 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.
  • the first body 100 may be composed of a plurality of side chambers 110.
  • 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 central chamber 310 and the upper part of the side chamber 110 (see FIGS. 10 and 11).
  • 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.
  • 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.
  • 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 10).
  • 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.
  • 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.
  • the inner lid 220 can be combined with the central chamber 310 to seal the upper part of the central chamber 310.
  • 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.
  • 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.
  • a syringe pump that can pressurize and depressurize using only internal air.
  • the lower reagent moving means 320 connects the side chamber 110 and the lower part of the central chamber 310 and serves as a passage for reagents.
  • 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.
  • the lower reagent moving means 320 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. 12).
  • 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.
  • 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.
  • the lower reagent moving means 320 may include a moving channel therein.
  • the moving channel it can be a passage through which the reagent moves.
  • 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.
  • the central chamber 310 may be connected to the center of the second body 300.
  • 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, and thus the lower part of each side chamber 110 can communicate with the lower reagent moving means 320 (see FIG. 12).
  • 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.
  • 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.
  • 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. 10 and 11).
  • 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. 11).
  • the second body 300 may be rotatably connected to the lower part of the first body 100.
  • sealing of the side chamber 110 installed in the first body 100 may be problematic.
  • 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.
  • 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. 11).
  • part or all of it may have a shape whose diameter narrows toward the bottom (see FIG. 13).
  • a supply hole 322 connected to the lower reagent moving means 320 may be formed in the lower center of the central chamber 310.
  • 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.
  • the first temperature control unit is a part that controls the overall temperature inside the system and may largely include a heater and an air circulation device (see Figure 1). Since the gene extraction and detection system of the present invention is intended to separate and analyze body fluids including blood used in the human body and causative bacteria contained therein, it is preferable to maintain a temperature of 30 to 50°C. Therefore, by using the first temperature control unit 20, the temperature of the survival system can be maintained at 30 to 50°C, preferably 35 to 40°C.
  • an air circulation device, a heater 21, and an induction duct 22 may be sequentially arranged to control the temperature by performing overall air circulation within the system.
  • the air sucked in using the air circulation device can be heated to a constant temperature while passing through the heater 21, and can be discharged through the induction duct 22 to circulate inside the system.
  • the induction duct 22 it may be installed in a direction where air continuously circulates inside the system, but preferably, the end of the induction duct 22 is installed toward the cartridge so that it is located near the cartridge. It is desirable to keep the temperature constant (see Figure 3).
  • the pump 30 is connected to the cartridge and can be used to pressurize or depressurize the cartridge central chamber.
  • the movement of fluid (reagent, etc.) inside the cartridge is performed using pressurization or depressurization of the central chamber, so the fluid inside the cartridge can be moved using the pump.
  • it is desirable to use a syringe pump because it must be possible to precisely control a large volume of 20ml or more and a small volume of solution of 50ul or less.
  • a tube connecting the pump and the cartridge is installed. may have an inner diameter of 2 mm or less.
  • the tube may be made of high hardness rubber, Tygon, urethane, Teflon, etc. (see Figure 2).
  • the fixing part 40 is used to fix the upper part of the cartridge 10 and is a part that fixes the upper part of the cartridge and fixes the outer lid and the first body at a desired position (FIGS. 2 and 4 reference).
  • the cartridge 10 of the present invention operates by rotating the second body 200 to connect the desired side chamber and the central chamber. That is, the other side of the lower reagent transfer device is connected to the lower part of each side chamber by adjusting the rotation angle. Therefore, in order to precisely control a small amount of solution of less than 50ul, the outlet hole formed at the bottom of each side chamber has a size of less than 2mm, so precise angle adjustment is necessary.
  • the cartridge 10 must be able to be fixed at an accurate angle in the equipment, and the rotating cartridge lower plate (second body) must be able to be fixed at an accurate angle to the rotating mechanism of the equipment.
  • an inclined fixing structure 250 is placed on the side of the cartridge, and a structure having an inclination of the same angle and direction is formed on the fixing part of the system to primarily adjust the angle of the cartridge.
  • horizontal direction fixation and vertical direction fixation can be performed.
  • the fixing structure 40 may have a plurality of pin-hole structures at the top, which can minimize the influence of errors that may occur due to injection deviation of the cartridge 10 ( 4).
  • the rotating part 50 is a part that rotates the lower part of the cartridge, that is, the second body 300, and connects the desired side chamber and the central chamber by rotating the second body 300.
  • the lower reagent moving means 320 requires a magnet to be selectively adhered or separated, and a direct heating process is required.
  • a hollow motor structure can be adopted, with the magnet and heater module positioned in the center and combined with the lower plate of the cartridge on the outside (see Figure 5).
  • the lower reagent moving means 320 of the present invention can be manufactured integrally with the second body 300, as seen above. Therefore, it is possible to rotate the lower reagent moving means 320 by rotating the second body 300. At this time, if the lower outer circumferential surface of the second body 300 is made not as a complete circle but as a circle with a portion cut off, it can be combined with the second body fixing part and rotated by a desired amount (see FIGS. 5 and 12).
  • the lower surface of the second body fixing part 51 is provided with a second temperature control part 70 and a magnetic body 60, as seen above, to provide heating and magnetic force when the lower reagent moving means reaches the desired position. It can be done.
  • the rotating part may be manufactured so that only the inner peripheral surface of the second body fixing part 51 rotates, and the bottom surface 52 of the second body fixing part does not rotate (see FIG. 5).
  • the second body fixing part may also have a pin-hole (53) structure to reduce the influence of cartridge injection deviation.
  • the magnetic body 60 is installed on the rotating part 50 and serves to fix the magnetic beads in the central chamber when the lower reagent moving means reaches a certain position.
  • the magnetic material 60 may be a permanent magnet made of metal, or it may also be a magnetic material made of an electromagnet to fix the magnetic bead at a desired point in time.
  • This magnetic material 60 must be able to collect or release magnetic beads depending on the rotation angle of the lower reagent moving means 520. Therefore, the magnetic body can be installed in the direction of the chamber where the reaction takes place with the magnetic beads fixed, and the magnet is not present in the direction of the chamber where the reaction takes place without the need for fixing the magnetic beads.
  • the second temperature control unit 70 is a part for controlling the temperature inside the lower reagent moving means 320 of the cartridge.
  • the reaction is mostly carried out at 30 to 50 °C, which is the temperature inside the system, but the hemolysis process requires a heating time of 1 to 15 minutes at 80 to 100 °C. Therefore, by installing the second temperature control unit at the lower part of the second body fixing part 51, the interior of the central chamber can be heated when the lower reagent moving means 320 reaches a certain position (FIG. 5 reference).
  • the other side of the lower reagent moving means is rotated so as not to be connected to the side chamber so that the extraction solution in the lower reagent moving means does not evaporate.
  • the third temperature control unit 80 is a part that controls the temperature of the PCR channel 400. Generally, in the case of PCR, the temperature is repeatedly raised and lowered, so in the case of the present invention, it is preferable to install a separate third temperature control unit 80 in the PCR channel to accurately control the temperature for each step. .
  • the rise and fall of the hondo must be performed within a short time, so it is preferable to use a plurality of Peltier elements 81.
  • the Peltier device is a device manufactured by joining two semiconductors and refers to a device that transfers heat from a semiconductor device on one side to a semiconductor device on the other side when electricity is supplied. In the case of such a Peltier device, heating and cooling can be repeatedly performed by changing the polarity of the supplied power, so it can be used to control the temperature of the PCR channel of the present invention, which requires repeated heating and cooling.
  • the Peltier element 81 can be installed above and below the PCR channel 400, and on the side connected to the PCR channel, there is a heat shield that protects the Peltier element from impact/wear and transfers the heat of the Peltier element to the PCR channel.
  • Highly conductive metal or polymer 82 can be installed.
  • An insulating material (83) can be installed on the side of the Peltier element (81) to reduce heat loss, and a heat exchanger (heatsink, 84) is installed on the opposite side of the Peltier element (61) to discharge the heat generated from the Peltier element.
  • a sensor can be installed to monitor the temperature of the metal or polymer 82 in direct contact with the PCR channel 400 of the cartridge, and the output of the Peltier element 81 can be adjusted using the temperature measured by the sensor as feedback. You can.
  • the upper Peltier element which is relatively easy to dissipate heat, can perform rapid heating/cooling in accordance with the denaturation, annealing, and elongation processes of nucleic acids
  • the lower Peltier element which is relatively difficult to dissipate heat, can perform heating/cooling quickly.
  • a detection unit 90 including an optical module may be installed in the PCR channel 400.
  • the third temperature control unit 80 and the optical module 90 are installed on a linear stage and can be selectively coupled to the PCR channel (see Figure 1 and Figure 2).
  • the third temperature control unit 80 and the optical module 90 may be installed in parallel on one moving axis. That is, the third temperature control unit 80 and the optical module 90 are installed in parallel to move linearly, and can be selectively coupled to the PCR channel 400 by moving them laterally.
  • the linear stage can set coordinates according to the number of channels to be imaged and the type of phosphor within the optical module, in addition to switching between the third temperature control unit 80 and the optical module 90.
  • LEDs of each wavelength band are attached to the LED PCB (92) to emit light according to signals, and the light emitted from the LED passes through the light guide structure (91) and an excitation filter. ) and passing through the lens module 93, the fluorescence of the cartridge PCR channel can be excited.
  • the fluorescence generated in the PCR channel passes through the emission filter and lens module 94 again through the light guide structure 95 and is converted into an electrical signal through the photodiode located on the photodiode PCB 96. can be converted.
  • the photodiode generates an electrical signal only for light having a certain wavelength, so when fluorescence is generated from a desired gene in the PCR channel, it is possible to detect it through the photodiode.
  • the gene extraction and detection system includes a blocking plate that spatially separates the upper and lower parts
  • the first temperature control unit, the fixing unit, the third temperature control unit, and the detection unit include the blocking plate.
  • the pump, the rotating part, and the magnetic body may be located below the blocking plate.
  • the temperature control unit that generates heat and the parts that need to maintain the heat generation state are located at the upper part centered on the blocking plate (15), and the motor and controller parts that are weak to heat are located in the lower part of the blocking plate (15).
  • the temperature is maintained at 30 to 50 °C by the first temperature control unit, and in the case of the nucleic acid elution process, the temperature must be maintained at 60 to 120 °C, so a heat blocking plate is used as described above.
  • thermal efficiency can be increased and equipment stability can be increased (see Figures 1 and 2).
  • the present invention also provides a cartridge in which a plurality of side chambers are arranged outside the central chamber and includes a PCR channel on one upper side; A first temperature control unit that maintains the space where the cartridge is installed at a constant temperature; a pump connected to the upper part of the cartridge to pressurize and depressurize the central chamber; A fixing part that secures the upper part of the cartridge; a rotating part that rotates the lower part of the cartridge to communicate with the side chamber and the central chamber at a desired position; A magnetic material that secures the magnetic beads inside the cartridge; a second temperature control unit that controls the temperature of the reagent moving means at the bottom of the cartridge; A third temperature control unit that controls the temperature of the PCR channel; And a gene extraction and detection method using a gene extraction and detection system including a detection unit that detects the fluorescence of the PCR channel after PCR is completed, (a) connecting a sample tube containing a sample with one of the side chambers of the cartridge.
  • the interior of the system may be preheated before the steps to be described later are performed, and the first temperature controller may be used for this preheating.
  • the interior of the system can be maintained at 30 to 50°C, preferably 35 to 40°C, a temperature similar to that of the human body, thereby minimizing the death of causative bacteria contained in the sample and the causative bacteria and the causative bacteria-trapping protein. Liver capture efficiency can be maximized.
  • 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).
  • the injection chamber 111 which is one of 110.
  • step (b) the samples and reagents loaded in each side chamber 110 are moved to the central chamber 310 to react.
  • 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.
  • 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 magnetic material to the lower part of the cartridge and then pressurizing the central chamber 310 to discharge excess reagent; (vi) rotating the lower reagent moving means to connect to the cleaning chamber 310;
  • 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.
  • 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.
  • 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.
  • the second body 300 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.
  • 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.
  • a portion of the collection reagent can be supplied to the magnetic bead chamber 113 by pressurizing the central chamber 310.
  • 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.
  • 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.
  • 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.
  • step (ii) 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.
  • 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.
  • 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.
  • 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.
  • 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. It is also possible to repeat the above movement 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.
  • 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 then pressurizing the central chamber 310 to discharge excess reagent.
  • the magnetic beads may be fixed to the inside of the lower reagent moving means by the magnetic material.
  • 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.
  • the magnetic body in the case of the magnetic body, as described above, it can be arranged uniformly on the bottom surface of the rotating part, and it is preferable that the second body is rotated and arranged at a point where the lower reagent moving means is connected to the collection reagent chamber.
  • 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.
  • 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.
  • 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.
  • the above washing may be repeated 2 to 10 times.
  • the number of cleaning chambers 114 may be 2 to 10, preferably 2 to 5.
  • 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.
  • 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.
  • 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).
  • 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.
  • a second temperature control means for performing heating as described above may be installed on the bottom surface of the rotating part in the direction of the nucleic acid elution chamber.
  • the lower reagent transport means can be heated when the lower reagent transport means is connected to the nucleic acid elution chamber.
  • step (b) movement between the side chamber and the central chamber in step (b) can be performed using the pump.
  • the pump may be connected to the central chamber through a pump connection at the top of the central chamber. Through this, the pump can pressurize or depressurize the central chamber, and according to this pressurization or depressurization, water, etc. can flow into or out of the central chamber.
  • RNA or DNA mixed with a buffer for nucleic acid elution may exist inside the central chamber 310.
  • PCR can be performed by pressurizing the central chamber 310 and transferring the extracted gene to the PCR channel 400 (step (d)).
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne une cartouche permettant une extraction et une détection d'un gène, pouvant sélectionner un antibiotique approprié pour un patient à un stade précoce grâce à l'identification de bactéries ou de souches provoquant une infection et à de multiples informations de détection d'un gène de résistance aux antibiotiques. La présente invention concerne un système d'extraction et de détection d'un gène. Le système comprend : une cartouche dans laquelle une pluralité de chambres latérales sont disposées à l'extérieur d'une chambre centrale et un canal de PCR est incorporé au niveau d'un côté supérieur; une première unité de régulation de température qui maintient à une température constante un espace dans lequel la cartouche est installée; une pompe qui est reliée à une partie supérieure de la cartouche et qui pressurise et dépressurise la chambre centrale; une partie de fixation qui fixe la partie supérieure de la cartouche; une partie rotative qui fait tourner une partie inférieure de la cartouche et permet aux chambres latérales et à la chambre centrale de communiquer en une position souhaitée; un matériau magnétique qui fixe des billes magnétiques internes de la cartouche; une deuxième unité de régulation de température qui régule la température de la chambre centrale; une troisième unité de régulation de température qui régule la température du canal de PCR; et une partie de détection qui détecte la fluorescence du canal de PCR après la fin de la PCR.
PCT/KR2022/019180 2022-11-30 2022-11-30 Système d'extraction et de détection d'un gène WO2024117299A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016117726A1 (fr) * 2015-01-23 2016-07-28 Infopia Co., Ltd. Cartouche
KR20180048558A (ko) * 2015-06-05 2018-05-10 아반바이오 인코포레이티드 생물학적 샘플로부터 바이오분자를 정제하고 테스트하는 디바이스의 구성요소, 디바이스 및 방법
KR20190018895A (ko) * 2017-08-16 2019-02-26 (주)오상헬스케어 유전자 분석 장치용 카트리지 및 이를 포함하는 유전자 분석 장치
KR20190059294A (ko) * 2016-09-23 2019-05-30 디엔에이넛지 리미티드 생물학적 샘플을 분석하기 위한 장치 및 방법.
WO2021138210A1 (fr) * 2019-12-30 2021-07-08 Abbott Diagnostics Scarborough, Inc. Dispositif de préparation d'échantillon et ses procédés d'utilisation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016117726A1 (fr) * 2015-01-23 2016-07-28 Infopia Co., Ltd. Cartouche
KR20180048558A (ko) * 2015-06-05 2018-05-10 아반바이오 인코포레이티드 생물학적 샘플로부터 바이오분자를 정제하고 테스트하는 디바이스의 구성요소, 디바이스 및 방법
KR20190059294A (ko) * 2016-09-23 2019-05-30 디엔에이넛지 리미티드 생물학적 샘플을 분석하기 위한 장치 및 방법.
KR20190018895A (ko) * 2017-08-16 2019-02-26 (주)오상헬스케어 유전자 분석 장치용 카트리지 및 이를 포함하는 유전자 분석 장치
WO2021138210A1 (fr) * 2019-12-30 2021-07-08 Abbott Diagnostics Scarborough, Inc. Dispositif de préparation d'échantillon et ses procédés d'utilisation

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