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WO2017131493A1 - Dispositif microfluidique pour détecter un gène mutant cible, et procédé pour améliorer l'efficacité de détection du dispositif microfluidique pour détecter un gène cible - Google Patents

Dispositif microfluidique pour détecter un gène mutant cible, et procédé pour améliorer l'efficacité de détection du dispositif microfluidique pour détecter un gène cible Download PDF

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WO2017131493A1
WO2017131493A1 PCT/KR2017/000998 KR2017000998W WO2017131493A1 WO 2017131493 A1 WO2017131493 A1 WO 2017131493A1 KR 2017000998 W KR2017000998 W KR 2017000998W WO 2017131493 A1 WO2017131493 A1 WO 2017131493A1
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gene
target
microfluidic device
detecting
primer
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Korean (ko)
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이혁진
정일영
김지수
최보람
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Ewha Womans University
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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
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/125—Rolling circle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a microfluidic device for detecting a target mutant gene, and a method for improving the detection efficiency of the microfluidic device for detecting a target gene.
  • a microfluidic device is a device in which each component is connected through a microfluidic channel on a substrate, and when a sample solution is injected, the entire process necessary for obtaining a detection signal is automated. It can be detected in a small amount of sample, uses less reagent, and the entire process is automated, which is convenient.
  • Publication No. 10-2014-0032094 discloses a microfluidic device that can be combined with a DNA template.
  • the microfluidic device disclosed in the above document has a two-layer structure, and the manufacturing process and the use method are complicated because the sample flows by controlling the angle of the flow path.
  • the pump is used to inject the sample, it must be externally powered and only one substance can be detected at a time.
  • Korean Patent Publication No. 10-2012-0125164 Name of the invention: EFFFR exon 19 polymorphism detection test oligonucleotide and use thereof)
  • the present invention is to provide a microfluidic device for detecting a target mutant gene and a detection method using the same.
  • the present invention also provides a microfluidic device kit for detecting a target gene with improved detection efficiency and a method for detecting a target gene using the same.
  • the present invention is a.
  • a first flow passage connected with the inlet to accommodate the sample solution introduced
  • a second flow path connected to the first flow path
  • a microfluidic device for detecting a target mutant gene comprising an outlet connected to the second flow path
  • the second flow path surface is coated
  • a primer is immobilized on the coating
  • the fixed primer is coupled to a template for detecting a target mutant gene
  • the target mutant gene detection template includes a primer binding portion that complementarily binds to the primer, a binding site complementary to the first template bound to one end of the primer binding portion-a first target mutation gene binding site, and the primer binding Provided is a microfluidic device for detecting a target mutant gene, comprising a complementary binding site-second target mutant gene binding site in a second template bound to the other end of the moiety.
  • the second flow path may be 1 to 20.
  • the second flow path is 2 to 20, each of the second flow path is branched at the end of the first flow path, the target mutant gene bound to the primer immobilized on each second flow path
  • the detection template may bind to the same or different mutant genes.
  • the coating is 5-hydroxydopamine hydrochloric acid, norepinephrine, epinephrine, pyrogallolamine, DOPA (3,4-Dihydroxyphenylalanine), catechin, tannins, pyrogallol, pyrocatechol , Heparin-catechol, chitosan-catechol, polyethyleneglycol-catechol, polyethyleneimine-catechol, polymethylmethacrylate-catechol, hyaluronic acid-catechol, polylysine-catechol, and It may be selected from the group containing polylysine (polylysine).
  • the primer is thiol, amine, hydroxyl, carboxyl, isothiocyanate, NHS ester, aldehyde, epoxide, carbonate, HOBt ester, glutaraldehyde, carbamate, imidazole carbamate, Terminals may be modified at least one selected from the group comprising maleimide, aziridine, sulfone, vinylsulfone, hydrazine, phenyl azide, benzophenone, anthraquinone, and diene groups.
  • the coating is 5-hydroxydopamine hydrochloric acid
  • the primer may be a terminal modified with a thiol or amine group.
  • the primer binding portion of the target mutant gene detection template may comprise a nucleotide sequence described in SEQ ID NO: 2.
  • mutant gene that can be detected by the microfluidic device for detecting a target mutant gene of the present invention.
  • the present invention can be applied to various mutant genes known in the art.
  • the mutant gene may be a cancer specific mutant gene appearing in cancer.
  • the cancer specific mutant gene may be a website such as http://www.mycancergenome.org, and any cancer specific mutant gene known in the art.
  • the mutant gene may be a carcinogenic mutant gene, which is a gene causing cancer, specifically a lung cancer specific mutant gene, more specifically, a gene in which EGFR exon 19 deletion has occurred, It is not limited.
  • Mutant genes that can be detected by the present invention are not limited to mutant genes associated with cancer, and other mutant genes associated with other congenital or acquired diseases or malformations may be used.
  • a mutant gene that can be detected by the present invention can be any mutant gene in which the normal gene and one or more bases are substituted, deleted or added.
  • the mutant gene may be a mutant gene in which two or more bases are substituted, deleted or added continuously or discontinuously.
  • the first target mutant gene binding site and the second target mutant gene binding site of the target mutant gene detection template of the present invention may be designed to bind complementarily to the mutant gene but not to the normal gene.
  • the mutant gene of the present invention can be, for example, a mutation of an epidermal growth factor receptor (EGFR) gene.
  • the mutant gene may be a gene in which EGFR exon 19 deletion (hereinafter, 'EGFR 19del') occurs.
  • EGFR 19del is a deletion of several to several ten bases consecutively in exon 19 of the EGFR gene, and a plurality of variants are known. Exemplary variants of EGFR 19del are shown in Table 1 below. In the table below, '-' means a deletion, lowercase means a mutation, and 'wild type' means a gene of the normal EGFR exon 19 part without deletion. Korean Patent Publication No. 10-2012-0125164 describes variously about the EGFR exon 19 polymorph, and many other documents have been reported about the EGFR exon 19 polymorph. However, EGFR 19del is not limited to that described in Table 1 below.
  • the EGFR 19del is a mutation commonly occurring in lung cancers such as Non-Small Cell Lung Cancer (NSCLC), and among them, abundantly observed in adenocarcinoma. Whether EGFR 19del has occurred can be used to confirm lung cancer.
  • NSCLC Non-Small Cell Lung Cancer
  • iresa compound name: gefitinib
  • tarseva compound name: erlotinib
  • geotripps compound name: afatinib
  • Numerous target anticancer agents for 19del's lung cancer are currently under development or clinical trials.
  • a mutant gene in which various variants of EGFR 19del can be detected quickly and accurately can be used to confirm lung cancer, and an appropriate anti-cancer agent can be selected to plan treatment of lung cancer patients. It can be a decisive help in establishing.
  • the present invention provides a method for producing a microfluidic device for detecting a target mutant gene, comprising the following steps.
  • a microfluidic device including a substrate, an inlet formed on the substrate, a first flow path connected to the inlet to receive the sample solution introduced therein, a second flow path connected to the first flow path, and an outlet connected to the second flow path Providing a;
  • the present invention provides a method for detecting a target mutant gene using the microfluidic device for detecting the target mutant gene comprising the following steps.
  • the steps c) and d) may be performed simultaneously (i.e., let the ligation and the rotation amplification reaction occur simultaneously), or c) after step d) is performed ( That is, after the ligation to cause a rotation ring amplification reaction). Simultaneously or separately, steps c) and d) may be determined according to the number and type of target substances to be detected.
  • the method is
  • the microfluidic device for detecting a target mutant gene of the present invention can be used for detection of various mutant genes occurring in humans and animals.
  • the animal includes all mammals, birds, reptiles, amphibians, fish and the like.
  • the present invention provides a microfluidic device for detecting a target gene
  • microfluidic device kit for detecting a target gene, comprising additional primers.
  • the microfluidic device is a microfluidic device.
  • a first flow passage connected with the inlet to accommodate the sample solution introduced
  • a second flow path connected to the first flow path
  • a microfluidic device for detecting a target gene comprising an outlet connected to the second flow path
  • the second flow path surface is coated
  • a primer is immobilized on the coating
  • the fixed primer is coupled to a template for detecting a target gene
  • the target gene detection template includes a primer binding portion that complementarily binds to the primer, a binding site complementary to the first template bound to one end of the primer binding portion-a first target gene binding portion, and the other of the primer binding portion.
  • a microfluidic device for detecting target genes comprising a complementary binding site-second target gene binding site in a second template bound to a terminal, and the specific configuration and features of the microfluidic device for detecting target genes are described in Application No. 10. -2015-0151941 (name of the invention: a microfluidic device for detecting a target gene, a preparation method thereof and a detection method using the same).
  • the additional primer may be the entire sequence, or a partial sequence of the primer binding portion of the target gene detection template, for example 5 to 50, preferably 10 to 25 Some, more preferably 15 to 22 bases in length.
  • the additional primer sequence is the same sequence as the sequence, or a part of the sequence. It may be a corresponding sequence, and in the case of a sequence corresponding to a part of the sequence, it may be preferably a sequence consisting of 10 to 25 bases, such as SEQ ID NO: 3 (5 ′ TGC TAG TAT C 3 ′).
  • the ligase may be a DNA ligase, such as T7 ligase, or CircLigase TM.
  • the isothermal nucleic acid polymerase may be phi 29 polymerase.
  • the kit may further include dithiothreitol (DTT) or pyrophosphatase.
  • DTT dithiothreitol
  • pyrophosphatase pyrophosphatase
  • the kit may further include a dyeing reagent, a high salt solution, or a fluorescent reagent.
  • the present invention provides a method for producing a microfluidic device comprising: a) providing a microfluidic device for detecting a target gene;
  • additional primers are coupled in the middle of the sequence to be rotated and amplified by the addition of additional primers, thereby causing amplification of the template in a larger amount more rapidly. That is, the addition of additional primers significantly shortens the time for amplification of the nucleic acid mass and can be detected much faster, and also if the target contains a small amount of the target gene or if the amount of the sample itself is small.
  • By increasing the amplification site through the second amplification by a large amount of template amplification can form a large nucleic acid clumps, thereby increasing the accuracy of detection.
  • the target gene detection method of the present invention is a daughter strand generated through the second amplification (additional amplification) by adding additional primers (mother strand generated by the first amplification (mother) Complementary binding to the strand) forms a DNA hydrogel faster and more robustly, thus improving the detection efficiency compared to the conventional detection method.
  • the method of detecting a target gene using a microfluidic device kit for detecting a target gene including the additional primer is used for detecting genes that can be derived from all origins such as animals, plants, bacteria, viruses, fungi, and the environment. Applicable
  • the target gene to be detected may be a pathogen gene.
  • the target gene can be targeted to any pathogen that knows the nucleic acid sequence.
  • the pathogen is avian influenza, SARS, Escherichia coli O157: H7, Mycobacterium tuberculosis, Bacillus anthracis, Streptococcus pneumonia, Malaria (Plasmodium), Salmonella (Salmonella), Hepatitis A, B, C, D and E viruses, Françasella tularensis, Yersinia pestis, Ersinia enterocolitica and Ebola virus, It is useful for the detection of MERS-Cov virus.
  • the pathogen is also useful for the detection of pneumococci, enterococci, staphylococcus, tropic fever and pathogens with antibiotic resistance such as tuberculosis or malaria bacteria in the third world.
  • the target gene to be detected may be a mutant gene.
  • the mutant gene may be a cancer specific mutant gene that appears in cancer.
  • the cancer specific mutant gene may be any cancer specific mutant gene known in the art, and specific cancer specific mutant genes are as described above. More specifically, it may be a lung cancer specific mutant gene, such as but not limited to a gene in which EGFR exon 19 deletion has occurred.
  • the present invention does not need to change the temperature for nucleic acid amplification, and can detect a target mutant gene without external power supply. Therefore, there is no need for complicated equipment, it is simple and inexpensive to manufacture, easy to operate, and easy to carry. It can also be used to detect a variety of mutant genes, and can be used to detect two or more mutant genes simultaneously.
  • the microfluidic device kit for detecting a target gene comprising the additional primer of the present invention; And by using the detection method using the same it can significantly shorten the detection time of the conventional microfluidic device for detecting the target material and increase the accuracy of the detection.
  • FIG. 1 is a diagram showing a result of detecting a gene having a EGFR exon 19 deletion mutation of SEQ ID NO: 5 using a microfluidic device for detecting a target mutant gene of the present invention with fluorescence.
  • Figure 2 is a schematic diagram showing that the secondary amplification of the ring by the addition of the additional primer can be amplified more quickly and a lot of templates.
  • FIG. 3 is a result of detecting a target gene by a method developed by the present inventor (named DNA Hydrogel Formation by Isothermal Amplification of Complementary Target in Fluidic Channels) using the same microfluidic device for detecting a target gene.
  • Figures (a) and (c)) and the results of detecting target genes (DbITACT-TR) by adding additional primers (DhITACT-TR) developed this time are shown respectively (b) and (d).
  • FIG. 4 is a diagram showing an Atomic Force Microscope (AFM) image of a target mers coronavirus gene hydrogel formed through DhITACT.
  • AFM Atomic Force Microscope
  • FIG. 5 shows an Atomic Force Microscope (AFM) image of a target mers coronavirus gene hydrogel formed through DhITACT-TR.
  • AFM Atomic Force Microscope
  • FIG. 6 is a diagram showing the results of rheology measurement of target mers coronavirus gene hydrogel formed through DhITACT and DhITACT-TR.
  • Figure 7 is a diagram confirming the result of detecting the gene for which the EGFR exon 19 deletion mutation of SEQ ID NO: 5 by the target gene detection method of the present invention with the addition of additional primers.
  • target mutant gene that can be detected by the microfluidic device for detecting a target mutant gene of the present invention is not limited, and may be any mutant gene having one or more base differences from a normal gene.
  • Mutant genes that can be detected with the present invention can be any mutant genes associated with a congenital or acquired disease or malformation.
  • the mutant gene may be a mutant gene in which two or more genes are substituted or deleted continuously or discontinuously.
  • the mutant gene may be a gene described by SEQ ID NO: 5, which is a kind of EGFR 19del. Comparing the mutant gene with a normal gene is shown in Table 2 below.
  • the template for detecting the target mutant gene of the present invention can be designed as follows (SEQ ID NO: 6).
  • the first target mutant gene binding site and the second target mutant gene binding site shown in bold, bind complementarily to 'AAATTCCCGGGA' and 'ATTAAGAGAAGCCAACAAGG' in the mutant gene of SEQ ID NO: 5, respectively. That is, the template can be designed such that the target mutant gene binding site complementarily binds only to the mutant gene.
  • the dark gray portion is a 'primer binding portion', and is prepared to bind complementarily with the primer represented by SEQ ID NO: 1 (5′-Thiol-AAA AAA AAA GGG ACG TCG ATA CTA GCA TGC TA-3 ′). (SEQ ID NO: 2).
  • the light gray portions are portions that complementarily bind to each other in the template (complementary binding sites in the first template and complementary binding sites in the second template).
  • the method for detecting a target gene using the microfluidic device for detecting a target substance that is, the microfluidic device for detecting a target gene, invented by the inventors of the present invention is disclosed in Korean Patent Application No. 10-2015-0151941 (name of the invention: for detecting a target gene).
  • the additional primer may be the entire sequence or a partial sequence of the primer binding portion of the target gene detection template used in the microfluidic device, for example, a partial sequence of 10 to 25 bases in length.
  • the upper two diagrams show a process in which rotation amplification occurs in the method of the existing application
  • the lower diagram shows additional primers coupled in the middle of the sequence to be amplified by the rotation when additional primers are added.
  • the process of amplifying the template in larger amounts is shown. That is, the addition of additional primers significantly shortens the time for amplification of the nucleic acid mass and can be detected much faster, and also template amplification when small amounts of target material are included in the sample or when the amount of the sample itself is small. This can occur a lot and form a large nucleic acid lump, which increases the accuracy of detection.
  • Example 1 Detection of EGFR 19del using microfluidic device for detecting target gene (cancer mutant gene)
  • 5-hydroxydopamine hydrochloric acid (5-hydroxydopamine HCl) was used as the coating material of the second flow path. Specifically, 5-hydroxydopamine hydrochloric acid (Sigma Aldrich) was dissolved in 10 mM Tris buffer (1M UltraPure 1M Tris-HCl pH 8.0 / Invitrogen) at a concentration of 1 mg / ml. Then, the pH was adjusted to 8 to prepare a coating composition. Wherein in the coating composition for a 1 ⁇ -Slide III 3in1 uncoated Microscopy Chamber filled the each second flow path of the product. After 2 hours it was washed with DDW (Water Purification System / LABOGENE).
  • DDW Water Purification System / LABOGENE
  • Primer was used to purchase Primer-5SS-polyA9 (BIONEER) having the following sequence.
  • the 5 'end of the primer sequence is attached to a thiol group.
  • the primer composition was prepared by the addition. Allow 4 hours to break any disulfide bonds that may have been generated in the primer by DTT, and then at 132,000 rpm, 4 ° C., 25 minutes using a 3K amicon tube (Amicon Ultra Centrifugal Filters 3K / MILLIPORE). First centrifugation, 40 ⁇ l DDW was added and second centrifugation to completely remove DTT remaining after reaction.
  • SEQ ID NO: 6 the template for detecting EGFR 19del described in SEQ ID NO: 5 (SEQ ID NO: 6) was prepared as follows.
  • the EGFR 19del was detected by DDW (Water Purification System / LABOGENE) and then left for 2 hours to allow complementary binding to occur between the primer binding portion of the template for detection and the second immobilized primer.
  • the right channel contains a template for detection of EGFR 19del in which nicking has already occurred (positive control).
  • a solution containing the EGFR 19del gene of SEQ ID NO: 5 was prepared as a sample solution.
  • a solution containing the EGFR normal gene of SEQ ID NO: 4 was prepared.
  • 3.5 ⁇ l of the sample solution was introduced into the second channel in the middle, and the negative control solution was introduced into the second channel on the left.
  • 30 ⁇ l of T7 ligase, 0.2 ⁇ l of 100 mM DTT, and 24.8 ⁇ l of Water Purification System / LABOGENE (DDW) were used to fill the second flow path of the microfluidic device for target gene detection. It was placed in a plastic container sealed with a para film to prevent the moisture in the second flow passage.
  • the plastics were filled with a tissue dampened with water and water at 30 ° C. Thereafter, it was left to stand at a non-shaking, 30 ° C., and 3 hours to allow the ligation of nicks of the EGFR 19del detection template.
  • the EGFR 19del detection template was amplified in the central flow path of FIG. 1 and entangled with hydroxydopamine hydrochloric acid coated with the second flow path to form a large hydrogel mass to block the outlet of the second flow path. there was. Similarly, it was confirmed that the outlet of the second flow path was blocked in the right flow path as the positive control. In contrast, no fluorescence was observed in the left channel, which was a negative control.
  • the inventors have invented a microfluidic device for detecting a target gene.
  • the present inventors invented a method of shortening the detection time of the microfluidic device and increasing the accuracy as follows.
  • a microfluidic device for detecting a target MERS gene was prepared as described in Experiment 5 of Application No. 10-2015-0151941.
  • the basic structure of the device is as described in 1-1 above, and the template sequence is as follows.
  • portions complementary to the target mers coronavirus gene are shown in bold letters, and portions complementary to the primers are light gray on a light gray background, and the portions complementary to the primer are displayed in dark gray. It was.
  • target mers coronavirus gene sequence to which the first target gene binding site and the second target gene binding site of the template sequence bind complementarily is as follows.
  • the sample solution was placed in the second channel in the middle and the negative control solution was placed in the leftmost second channel in the same manner as in the method of 1-2. Inflow.
  • 30 ⁇ l of T7 ligase, 0.2 ⁇ l of 100 mM DTT, and 24.8 ⁇ l of Water Purification System / LABOGENE (DDW) were used to fill the second flow path of the microfluidic device for target gene detection. It was placed in a plastic container sealed with a para film to prevent the moisture in the second flow passage. The plastics were filled with a tissue dampened with water and water at 30 ° C. Thereafter, it was left to stand at a non-shaking, 30 DEG C, and 3 hours to allow the ligation of the nick of the template to occur.
  • a microfluidic device for detecting a target MERS gene was prepared, a sample solution containing the MERS virus gene was injected, and a ligation reaction and a rotation ring amplification reaction occurred in sequence.
  • 'additional primer' (consisting of a short DNA sequence) was further added during the rotation amplification reaction. Additional primer sequences added are as follows.
  • the present inventors also added the pore size of the hydrogel using an Atomic Force Microscope (AFM) and to confirm the improved detection efficiency of the microfluidic device for target gene detection by adding additional primers. Rheology was measured.
  • AFM Atomic Force Microscope
  • the target MERS gene hydrogel of Example 2-1 which was amplified by adding the target MERS gene hydrogel of Example 1 and an additional primer, which was formed by using an atomic force microscope (NX10, Park Systems, South Korea) as an existing device
  • NX10 Park Systems, South Korea
  • the pore size of the mass was measured and the results are shown in FIGS. 4 and 5.
  • the average pore size of the hydrogel amplified using the existing microfluidic device for detecting target genes was 56 nm (FIG. 4), but when additional primers were added (DhITACT -TR) average pore size was 16 nm (Fig. 5), the density was confirmed to increase about 3.5 times. That is, by adding additional primers to form a more robust DNA hydrogel mass it was confirmed that the detection efficiency was improved.
  • the MERS gene hydrogel (TR) formed through DhITACT-TR formed a more viscoelastic gel than the hydrogel (D) formed through DhITACT, and storage modulus (G ′) was also increased. High. That is, it was confirmed that by adding additional primers to form a hydrogel having a stronger elasticity, the detection efficiency of the microfluidic device was increased.
  • microfluidic device for detecting target mutant genes As described in 1-1 above, a microfluidic device for detecting EGFR 19del was prepared.
  • the specific template sequence (SEQ ID NO: 6) is as follows, and the meaning indicated in the sequence is the same as the MERS template.
  • target EGFR 19del gene sequence to which the first target gene binding site and the second target gene binding site of the template sequence bind complementarily is as follows.
  • amplification of the EGFR 19del gene was performed in the same manner as the method using the microfluidic device for detecting MERS, and an additional primer of SEQ ID NO: 3 was added.
  • the template was amplified to visually observe that the hydrogel mass was formed only 30 minutes after the reaction, and the time required for amplification was greatly shortened by the addition of additional primers. That is, the detection of the target EGFR 19del gene of Example 1-2 without adding the additional primer was able to visually confirm the hydrogel 2 hours after the rotation amplification reaction (Fig. 1), but the addition of the additional primer In this case, the time required for amplification of the EGFR 19del mutant gene was significantly shortened. Therefore, when the additional primer is added, even when a small amount of the target EGFR 19del mutant gene is present in the sample solution, it is possible to detect with high sensitivity, thereby increasing the accuracy.

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Abstract

La présente invention concerne un dispositif microfluidique pour détecter un gène mutant cible. Le dispositif de la présente invention ne nécessite pas de variations de température pour une amplification d'acide nucléique, et peut détecter un gène mutant cible même en l'absence de fourniture d'énergie externe. Par conséquent, le dispositif n'a pas besoin d'être accompagné d'un équipement compliqué, peut être fabriqué de manière simple et peu coûteuse, est pratique à utiliser et est facile à transporter. En outre, le dispositif peut trouver des applications dans la détection d'une variété de gènes mutants et la détection simultanée d'au moins deux gènes mutants. En outre, la présente invention concerne un procédé pour améliorer une efficacité de détection d'un dispositif microfluidique pour détecter un gène cible. Lorsqu'il est utilisé, le procédé de la présente invention peut réduire considérablement le temps nécessaire pour des dispositifs microfluidiques traditionnels pour détecter des gènes cibles, et peut augmenter la précision de détection.
PCT/KR2017/000998 2016-01-29 2017-01-26 Dispositif microfluidique pour détecter un gène mutant cible, et procédé pour améliorer l'efficacité de détection du dispositif microfluidique pour détecter un gène cible Ceased WO2017131493A1 (fr)

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