WO2018074614A1 - Distributeur quantitatif automatique, appareil d'extraction-amplification de gène de type continu comprenant le distributeur et procédé d'extraction-amplification de gène de type continu - Google Patents

Distributeur quantitatif automatique, appareil d'extraction-amplification de gène de type continu comprenant le distributeur et procédé d'extraction-amplification de gène de type continu Download PDF

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Publication number
WO2018074614A1
WO2018074614A1 PCT/KR2016/011649 KR2016011649W WO2018074614A1 WO 2018074614 A1 WO2018074614 A1 WO 2018074614A1 KR 2016011649 W KR2016011649 W KR 2016011649W WO 2018074614 A1 WO2018074614 A1 WO 2018074614A1
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WO
WIPO (PCT)
Prior art keywords
gene
bar magnet
amplification
sample
continuous
Prior art date
Application number
PCT/KR2016/011649
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English (en)
Korean (ko)
Inventor
유문조
송강희
신은심
Original Assignee
휴스텝스 주식회사
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
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Application filed by 휴스텝스 주식회사 filed Critical 휴스텝스 주식회사
Priority to PCT/KR2016/011649 priority Critical patent/WO2018074614A1/fr
Publication of WO2018074614A1 publication Critical patent/WO2018074614A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices

Definitions

  • the present invention relates to a one-step automatic test (or diagnostic) equipment in which the extraction and amplification of a gene can be performed continuously. More specifically, the aspirator extracts a certain amount of the genetic material extracted in the gene extraction step, and then the subsequent gene.
  • An automatic quantitative dispenser configured to transfer a certain amount of the genetic material into the dispensing container by transfer to the amplification step, a continuous gene extraction-amplification equipment including the automatic quantitative dispenser, and a gene using the equipment To extract-amplify.
  • Examples of the substance to be tested include DNA, a messenger RNA encoding a protein, proteins related to them, specific metabolites, and the like.
  • the purpose of genetic testing includes prediction of risk of developing a genetic disease, identification of carriers, and the like.
  • Genetic testing requires gene extraction and gene amplification, of which gene extraction begins with the sampling phase.
  • a cotton swab is rubbed on the skin of a chicken and a sample containing the genetic material of contaminants such as bacteria is buried into the swab, and then the swab is placed in a test tube containing a specific solution.
  • various foreign substances are dispersed in a specific solution together with a sample on a cotton swab.
  • proteins or phospholipids surrounding a cell nucleus are destroyed by a specific solution, and the gene in the nucleus is released into a specific solution. You lose.
  • Magnetic beads used in the collecting step are known in the art, and include a ball-shaped magnetic material (for example, ferrous metal), a plastic or glass coating layer surrounding the magnetic material, and a silica coating layer covering the coating layer.
  • the silica coating layer has a property of attracting and attracting genes.
  • the bar magnet (more specifically, the electromagnet)
  • the magnetic material in the magnetic bead is attracted by the magnetic force of the bar magnet, and the magnetic bead gathers and sticks around the bar magnet.
  • a washing step is performed to remove impurities (foreign substances) other than the genes adsorbed on the magnetic beads.
  • washing steps are typically carried out twice, but there is no limit to the number of runs.
  • the washing step is performed in a container (eg, a test tube) containing a washing buffer (washing liquid or washing liquid).
  • a bar magnet having a plurality of magnetic beads stuck to it is transferred from a previous collecting step to a washing step, and then the bar magnet is placed in a test tube containing a washing buffer, and a current is cut off. To eliminate the magnetic force of the electromagnet.
  • the magnetic magnet is re-magnetized by flowing an electric current through the bar magnet from which the magnetic force is removed, so that a plurality of magnetic beads to which a gene (genetic material) is adsorbed again stick to the bar magnet.
  • the bar magnet which has a plurality of magnetic beads attached thereto, is transferred to the second washing step, and as described in the first washing step, the magnetic force of the bar magnet is removed, and after a certain time, the magnetic force of the bar magnet is reset.
  • the second wash is performed by recovering.
  • This gene elution step is performed using an elution buffer (eluent) that prevents the gene from sticking to the magnetic beads.
  • elution buffer eluent
  • the magnetic beads adhering to the rod magnets are dispersed in the ion buffer by inserting the bar magnets transferred from the previous washing step into the gene eluting step into a test tube containing the illu- tion buffer and blocking the current.
  • Patent Document 2 gene extraction method in one step
  • Patent Document 4 automatic purification device, multi-well plate kit, and a method of extracting nucleic acids from biological samples.
  • Eluents containing only genes through each of the above steps are quantified using a pipette or a divider, and then injected into a PCR (Polymerase Chain Reaction) amplifier capable of temperature control, thereby generating gene growth (amplification).
  • PCR Polymerase Chain Reaction
  • Patent Document 1 gene amplification apparatus and its production method
  • Patent Document 3 non-contact heating gene amplification system
  • Patent Document 1 Publication No. 10-2015-0024050
  • Patent Document 2 Publication No. 2002-0029477
  • Patent Document 3 Registration No. 10-1302748
  • Patent Document 4 Registration No. 10-1025135
  • the present invention has been made to solve the problems of the conventional genetic test equipment, and provides an automatic quantitative divider capable of transplanting a certain amount of the Illution buffer containing the gene from the gene extraction device to the gene amplification device.
  • the purpose is to.
  • the continuous gene extraction-amplification equipment employs a permanent magnet as a component for attracting magnetic beads used for gene collection, so that the equipment is The purpose is to provide a miniaturized and portable equipment.
  • the present invention has another object to provide a method in which the extraction and amplification of the gene can be carried out continuously through a series of processes.
  • a volume of liquid is formed in response to a volume change portion formed at one end of the connecting end to which the external actuator can be coupled, and a volume change portion generated when the external actuator is driven while being coupled to the connecting end of the volume variable. And a pipette portion connected to the other end of the volume variable portion so as to be sucked or discharged, and a locking jaw formed on the outer circumferential surface of the pipette portion.
  • a sample kit seat where a sample kit capable of performing gene extraction through an automated series of steps is placed, a PCR amplifier for performing gene amplification, and a waste unit disposed between the sample kit seat and the PCR amplifier;
  • a continuous gene extraction-amplification device comprising a rod magnet moving rail for transferring a rod magnet from a sample kit seating part to a PCR amplifier according to a given processing recipe, wherein the sample kit is for fitting at the end of the rod magnet.
  • a pollution prevention tip storage unit in which the pollution prevention tip is stored;
  • a magnetic bead storage unit having a plurality of magnetic beads having magnetic beads that can stick to the rod magnets and silica coating layers to which the genes can be adsorbed;
  • a sample sample storage unit capable of injecting a liquid sample in which genes and inevitable impurities are mixed; Washing buffer storage for relatively high impurity concentration while increasing the gene concentration;
  • An illution buffer storage unit configured to store an illution buffer for containing only genes;
  • an automatic metering dispenser storage unit in which the metering dispenser is built in [1] is spaced apart from each other, and the disposal part includes an anti-fouling tip inserted at the end of the bar magnet and at the end of the bar magnet.
  • Continuous gene extraction-amplification equipment characterized in that consisting of a configuration formed on one side of the notch to enable separation (uncut).
  • a continuous gene extraction-amplification method characterized by automating all processes from gene extraction to gene amplification using the continuous gene extraction-amplification equipment described in [2] or [3].
  • the extraction and amplification of the gene can be automatically performed through a series of continuous processes.
  • the present invention is to overcome the difficulty of carrying a large volume by the components such as coils when using an existing electromagnet, using a permanent magnet to reduce the size to a small enough to carry a series of extraction and amplification while carrying in the field The process can be carried out efficiently.
  • FIG. 1 is a schematic configuration and use state of the automatic metering dispenser according to an embodiment of the present invention in chronological order, (a) is compressed by an external actuator in a state in which the metering dispenser is inserted into the test tube In this state, (b) shows the compression state in which the compression by the external actuator is performed in the state of (a), and (c) indicates that the external actuator moves in the opposite direction to the previous state in the state of (b).
  • the liquid in the test tube represents a fixed amount suction state in which a predetermined amount is sucked into the pipette part of the automatic metering dispenser. Departure state.
  • Figure 2 is a block diagram showing the schematic configuration of a continuous gene extraction-amplification equipment including an automatic quantitative divider according to an embodiment of the present invention.
  • FIG. 3 is a plan view illustrating an example of a sample kit in FIG. 2.
  • FIG. 4 is a view for explaining a process of continuously extracting and amplifying a gene using the continuous gene extraction-amplification equipment shown in FIG.
  • FIG. 5 is another diagram for describing a series of processes of continuously extracting and amplifying genes using the continuous gene extraction-amplification equipment shown in FIG. 2, and correspond to steps 3 to 6 of FIG. 4.
  • FIG. 1 illustrates a schematic configuration and use state of the automatic quantitative dispenser 100 according to an embodiment of the present invention in chronological order, (a) indicates that the automatic quantitative divider 100 is connected to the test tube 300. The reference state in which the compression by the external actuator is not performed in the inserted state is shown, and (b) indicates the compression state in which compression by the external actuator is performed in the state of (a).
  • (c) is a quantitative amount of the liquid 400 in the test tube 300 is sucked into the pipette portion 120 of the automatic quantitative dispenser 100 by operating the external actuator in the opposite direction in the state of (b) Indicated the suction state, (d) shows a departure state in which the automatic metering dispenser 100, which has sucked a certain amount of liquid 400, is separated from the test tube 300 and can be transferred to a subsequent step or a subsequent step.
  • the automatic metering dispenser 100 includes a volume variable part 110 having a connection end 111 to which an external actuator can be coupled at one end thereof, and an external actuator.
  • the volume variable portion 110 In response to the volume change amount of the volume variable portion 110 generated when it is driven in a state coupled to the connection end 111 of the volume variable portion 110, the volume variable portion 110 so as to suck or discharge a certain amount of liquid. It comprises a pipette 120 connected to the other end of the) and the locking step 130 formed on the outer peripheral surface of the pipette 120.
  • the volume variable part 110 has a bellows shape.
  • the volume (inner volume) decreases and when the external actuator moves away from the pipette part 120, the volume increases, and the pipette part 120 A predetermined amount of liquid is sucked in the c), or a predetermined amount of liquid is discharged from the pipette 120.
  • the locking jaw 130 formed on the outer circumferential surface of the pipette 120 is preferably an annular ring, but may be a form in which a part of the ring is cut.
  • the automatic metering dispenser 100 can be manufactured integrally by, for example, plastic injection molding.
  • the automatic quantitative divider 100 was developed for automatically transferring a certain amount of an illumination buffer containing a gene from a gene extraction device to a gene amplification device.
  • Other materials for example liquid chemicals, may be used for metered delivery.
  • the automatic metering dispenser 100 can be used wherever a quantitative transfer of liquid substance is required.
  • the automatic metering dispenser 100 is versatile and therefore, to emphasize that there is no limitation in the range of use, the external actuator for the drive element causing the volume change of the volume variable portion 110 is to be emphasized. It is given a comprehensive name, if limited to the field of genetic testing equipment, this external actuator corresponds to the bar magnet (200).
  • the bar magnet 200 is preferably composed of a permanent magnet.
  • FIG. 2 is a block diagram showing a schematic configuration of a continuous gene extraction-amplification apparatus according to an embodiment of the present invention
  • FIG. 3 is a plan view showing an example of a sample kit in FIG.
  • the continuous gene extraction-amplification equipment includes a sample in which a sample kit A1, in which a gene extraction can be performed through an automated series of processes, is placed.
  • the kit seating portion (A), the PCR amplifier (D) for performing gene amplification, the waste portion (C) disposed between the sample kit seating portion (A) and the PCR amplifier (D), and a given treatment recipe (operation method) It consists of a bar magnet moving rail (B) for transferring the bar magnet 200 toward the PCR amplifier (D) from the sample kit seating portion (A) according to the).
  • the rod magnet moving rail (B) is provided over the sample kit seating portion (A) and the PCR amplifier (D).
  • the bar magnet 200 is not only movable in the horizontal direction along the bar magnet moving rail (B), as shown in Figure 2, the sample kit seating portion (A) and waste (C) and the PCR amplifier ( It is comprised so that lifting up and down in the vertical direction toward D) is possible.
  • the sample kit A1 has a structure of 7 columns ⁇ 10 rows, and the number of rows may vary.
  • the sample kit A1 is formed in a row with a total of seven storage units spaced from each other, specifically, an anti-contamination tip storage unit in which an anti-contamination tip 500 for inserting at an end of the bar magnet 200 is stored.
  • a magnetic bead storage unit (a2) storing a plurality of magnetic beads having a silica coating layer capable of adsorbing magnetic material and genes that can stick to the rod magnet 200, and a liquid mixed with genes and unavoidable impurities
  • a sample sample storage unit (a3) capable of injecting a sample of the sample, two washing buffer storage units (a4 and a5) for relatively high impurity concentration and relatively high impurity concentration, and an illution buffer for containing only genes
  • the waste portion (C), as illustrated in Figure 4, the notch that allows the entry of the bar magnet 200 and the separation and removal of the contamination prevention tip 500 fitted to the end of the bar magnet 200 is one side. Consists of a configuration formed on.
  • the PCR amplifier D is provided above the amplifying unit D1, the lower heater D2 fixedly installed below the amplifying unit D1, and above the amplifying unit D1 to the amplifying unit D1. It consists of a movable upper heater D3, and the upper heater moving rail D4 in which the upper heater D3 is transportably installed. Since this configuration is known in the art, a detailed description thereof will be omitted.
  • FIG. 4 is a view illustrating a process of continuously extracting and amplifying a gene using a continuous gene extraction-amplification apparatus according to the present invention
  • FIG. 5 is a diagram corresponding to steps 3 to 6 of FIG.
  • the sample kit A1 illustrated in FIG. 3 is disposed on the sample kit seating portion A.
  • FIG. 3 is a diagrammatic representation of the sample kit seating portion A.
  • the bar magnet 200 is moved to just above the contamination prevention tip storage part a1 of the sample kit A1.
  • the anti-fouling tip 500 of the rubber material contained in the anti-fouling tip storage unit a1 is inserted into the end of the bar magnet 200 (1 step).
  • the bar magnet 200 When the anti-fouling tip 500 is covered on the end of the bar magnet 200, the bar magnet 200 is raised as it is, and then the bar magnet 200 is moved directly above the magnetic bead storage unit a2.
  • the magnetic bead 600 contained in the magnetic bead storage part a2 includes a ball-shaped magnetic body 610, a coating layer 620 of plastic or glass material surrounding the magnetic body 610. Since it is composed of a silica coating layer 630 surrounding the coating layer 620, when the end of the bar magnet 200 into the magnetic bead storage portion (a2), the plurality of magnetic beads stored in the magnetic bead storage portion (a2) Magnetic bead 600 is stuck to the bar magnet 200 (step 2).
  • a predetermined amount of liquid that is, a sample sample
  • a predetermined amount of liquid that is, a sample sample
  • the protein or phospholipid surrounding the cell nucleus is destroyed by a specific solution and the gene in the cell nucleus is released is injected into the sample sample storage unit a3 in advance.
  • the bar magnet 200 is moved to just above the washing buffer storage unit a4.
  • the bar magnet 200 When the bar magnet 200 is located directly above the washing buffer storage unit a4, the bar magnet 200 is lowered to lock the end of the bar magnet 200 to which the magnetic bead 600 is attached to the washing buffer. Vibration (shake or churning) in, to cause the impurities attached to the magnetic beads 600 with the gene to some extent to fall off (step 4).
  • step 5 After raising the bar magnet 200 to which the magnetic bead 600 is stuck, it moves to the second washing buffer storage unit a5 and repeats the same process as in step 4 (step 5). .
  • the bar magnet 200 to which the magnetic beads 600 are attached is moved to the upper portion of the illu- tion buffer storage a6, and then lowered to enter the illu- tion buffer storage a6, and the magnetic beads 600 are moved. While the portion of the bar magnet 200 is attached to the vibration buffer is applied to the vibration state, the gene is released from the surface of the magnetic bead 600, while the magnetic bead 600 itself is a bar magnet 200 Make sure they stick together (step 6).
  • the bar magnet 200 is moved to the place where the waste portion C is located, and then the rod magnet is lifted or the waste portion C is raised and lowered to prevent contamination of the rod magnet 200. Peel off tip 500.
  • the bar magnet 200 is moved immediately above the automatic metering dispenser storage unit a7 of the sample kit A1, and then lowered. It is coupled to the connection end 111 (step 8).
  • the bar magnet 200 to which the automatic quantitative divider 100 is coupled is moved upward and lowered to the illu- tion buffer storage unit a6, and then lowered. This sucks into the pipette 120 of the automatic metering dispenser 100.
  • the bar magnet 200 to which the automatic quantitative divider 100 is coupled is moved to the PCR amplifier D, while the upper heater D3 of the PCR amplifier D is moved to either side of the amplifier D1.
  • the pipette part 120 is inserted into the amplification part D1, and the volume variable part (s) into the bar magnet 200 is discharged from the pipette part 120 containing an amount of genes. Press 110).

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un distributeur quantitatif automatique qui comprend : une partie de changement de volume ayant une extrémité de liaison formée à une extrémité de cette dernière pour permettre le couplage d'un aimant droit à l'extrémité de liaison ; une partie pipette reliée à l'autre extrémité de la partie de changement de volume pour permettre l'introduction ou l'évacuation d'une quantité prédéterminée de liquide en réponse à une quantité de variation de volume de la partie de changement de volume ; et un seuil de mise en prise formé sur une surface circonférentielle externe de la partie pipette.
PCT/KR2016/011649 2016-10-17 2016-10-17 Distributeur quantitatif automatique, appareil d'extraction-amplification de gène de type continu comprenant le distributeur et procédé d'extraction-amplification de gène de type continu WO2018074614A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/KR2016/011649 WO2018074614A1 (fr) 2016-10-17 2016-10-17 Distributeur quantitatif automatique, appareil d'extraction-amplification de gène de type continu comprenant le distributeur et procédé d'extraction-amplification de gène de type continu

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2016/011649 WO2018074614A1 (fr) 2016-10-17 2016-10-17 Distributeur quantitatif automatique, appareil d'extraction-amplification de gène de type continu comprenant le distributeur et procédé d'extraction-amplification de gène de type continu

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WO2018074614A1 true WO2018074614A1 (fr) 2018-04-26

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR200189404Y1 (ko) * 2000-02-18 2000-07-15 황보현 자바라식 스포이드 튜브
KR20090019164A (ko) * 2007-08-20 2009-02-25 삼성전자주식회사 유전자 검출 방법 및 장치
JP5143570B2 (ja) * 2006-01-13 2013-02-13 ユニバーサル・バイオ・リサーチ株式会社 変形式分注チップ、変形式分注装置および変形式分注処理方法
KR20130092185A (ko) * 2012-02-10 2013-08-20 (주)바이오니아 생체시료의 자동 분석 장치 및 방법
KR101423936B1 (ko) * 2009-03-11 2014-07-29 (주)바이오니아 실시간 핵산 분석 통합 장치 및 이를 이용한 타겟 핵산의 검출방법
KR20160148201A (ko) * 2015-06-16 2016-12-26 휴스텝스 주식회사 자동 정량 분주기 및 상기 분주기를 포함하는 연속식 유전자 추출-증폭 장비와 연속식 유전자 추출-증폭 방법

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR200189404Y1 (ko) * 2000-02-18 2000-07-15 황보현 자바라식 스포이드 튜브
JP5143570B2 (ja) * 2006-01-13 2013-02-13 ユニバーサル・バイオ・リサーチ株式会社 変形式分注チップ、変形式分注装置および変形式分注処理方法
KR20090019164A (ko) * 2007-08-20 2009-02-25 삼성전자주식회사 유전자 검출 방법 및 장치
KR101423936B1 (ko) * 2009-03-11 2014-07-29 (주)바이오니아 실시간 핵산 분석 통합 장치 및 이를 이용한 타겟 핵산의 검출방법
KR20130092185A (ko) * 2012-02-10 2013-08-20 (주)바이오니아 생체시료의 자동 분석 장치 및 방법
KR20160148201A (ko) * 2015-06-16 2016-12-26 휴스텝스 주식회사 자동 정량 분주기 및 상기 분주기를 포함하는 연속식 유전자 추출-증폭 장비와 연속식 유전자 추출-증폭 방법

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