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WO2019009592A2 - Particule avec un matériau ucst appliqué à celle-ci, et procédé d'amplification d'acide nucléique l'utilisant - Google Patents

Particule avec un matériau ucst appliqué à celle-ci, et procédé d'amplification d'acide nucléique l'utilisant Download PDF

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Publication number
WO2019009592A2
WO2019009592A2 PCT/KR2018/007523 KR2018007523W WO2019009592A2 WO 2019009592 A2 WO2019009592 A2 WO 2019009592A2 KR 2018007523 W KR2018007523 W KR 2018007523W WO 2019009592 A2 WO2019009592 A2 WO 2019009592A2
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WIPO (PCT)
Prior art keywords
ucst
primer
nucleic acid
particles
hydrogel
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PCT/KR2018/007523
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English (en)
Korean (ko)
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WO2019009592A3 (fr
Inventor
김상경
김준선
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Korea Institute of Science and Technology KIST
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Korea Institute of Science and Technology KIST
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Priority claimed from KR1020180076483A external-priority patent/KR102088877B1/ko
Application filed by Korea Institute of Science and Technology KIST filed Critical Korea Institute of Science and Technology KIST
Priority to US16/628,346 priority Critical patent/US20200239944A1/en
Priority to CN201880057430.3A priority patent/CN111356771A/zh
Publication of WO2019009592A2 publication Critical patent/WO2019009592A2/fr
Publication of WO2019009592A3 publication Critical patent/WO2019009592A3/fr
Anticipated expiration legal-status Critical
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    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • a UCST particle containing a primer by introducing a new nucleic acid supply material, a hydrogel microparticle having a primer immobilized by including the UCST particle, and a nucleic acid amplification method and a nucleic acid amplification apparatus using the same are disclosed.
  • Solid-phase PCR is a technique for analyzing multiple target nucleic acids at the same time, using a primer or a probe immobilized on a solid surface.
  • PCR polymerase chain reaction
  • a UCST polymer matrix having an Upper Critical Solution Temperature (UCST) at 20 to 90 < 0 >C;
  • UCST Upper Critical Solution Temperature
  • PCR polymerase chain reaction
  • One aspect of the present invention is a PCR-use hydrogel microparticle in which at least one primer of the forward primer and the reverse primer of the target nucleic acid, or a probe of the target nucleic acid is immobilized as a polymerase chain reaction primer, and the hydrogel microparticles At least one primer of the omnidirectional primer and the reverse primer or a probe of a target nucleic acid is contained in the UCST particle to be fixed to the hydrogel fine particle,
  • the hydrogel microparticles provide a hydrogel microparticle, which is a porous structure comprising pores.
  • one aspect of the present invention provides a nucleic acid amplification apparatus comprising at least one UCST particle.
  • one aspect of the present invention provides a nucleic acid amplification apparatus comprising at least one of the hydrogel microparticles.
  • one aspect of the present invention is to prepare a UCST particle by adding at least one primer of an omnidirectional primer and a reverse primer of a target nucleic acid or a probe of a target nucleic acid to a liquid UCST (Upper Critical Solution Temperature)
  • the method comprising the steps of:
  • one aspect of the present invention is to prepare a UCST particle by adding at least one primer of an omnidirectional primer and a reverse primer of a target nucleic acid or a probe of a target nucleic acid to a liquid UCST (Upper Critical Solution Temperature) step;
  • the prepared UCST particles, a hydrogel monomer and a photoinitiator are mixed to prepare a prepolymer solution.
  • the solution is mixed with the omnidirectional primer And a primer that is not included in the UCST among the reverse primers; And discharging the prepolymer solution in a droplet form and curing the prepolymer solution to prepare hydrogel microparticles.
  • the present invention also provides a method for producing hydrogel microparticles.
  • a method of preparing a UCST particle comprising: injecting a UCST particle into a reaction chamber; Injecting a solution containing one or more target nucleic acids into the reaction chamber; And amplifying the target nucleic acid by polymerase chain reaction (PCR) of the target nucleic acid.
  • PCR polymerase chain reaction
  • a nucleic acid amplification method comprising: injecting at least one hydrogel fine particle into a reaction chamber; Injecting a solution containing one or more target nucleic acids into the reaction chamber; And amplifying the target nucleic acid by a polymerase chain reaction with the target nucleic acid.
  • the UCST particles of the present invention include one or more primers or probes of an omnidirectional and reverse primer, so that the primer and / or the probe are all treated with UCST particles until the denaturation step in the PCR, in particular, the one-step reverse transcription PCR It is possible to prevent the formation of a primer dimer due to non-specific binding between the primer and the primer or between the primer and the probe.
  • the hydrogel fine particles containing the UCST particles of the present invention can be prepared by fixing both the bi-directional primer and / or the probe to the hydrogel fine particle to supply the primer and / or probe necessary for the amplification reaction of the target nucleic acid to the corresponding particle, Generation of a duplex can be prevented.
  • the primer contained in the UCST particles flows out into the hydrogel microparticles in the denaturation step during the PCR process, the primer has a high degree of freedom.
  • the primer In the nucleic acid amplification, only one directional primer is fixed to the hydrogel microparticles, Can be shown to have the same high amplification efficiency as that put into solution.
  • FIG. 1 is a schematic diagram illustrating a principle of performing one-step reverse transcription (qPCR) using UCST particles containing reverse primer as one embodiment of the present invention. to be.
  • FIG. 2 is a diagram showing a UCST particle including a reverse primer and a probe as one embodiment of the present invention.
  • FIG. 2 is a diagram showing a UCST particle including a reverse primer and a probe as one embodiment of the present invention.
  • FIG. 3 is a graph showing an example of the present invention in which the hydrogel microparticles contain UCST particles and the UCST particles are melted at a temperature of UCST or higher in the PCR to flow out the primers contained in the UCST particles, Fig.
  • FIGS. 4A and 4B are graphs showing fluorescence signals obtained after one day, two days, and six days after the preparation of the UCST particles according to an embodiment of the present invention, as a result of performing one step reverse quantitative PCR.
  • the y-axis in FIG. 4A represents the absolute values of the respective fluorescence signals immediately after the production of the UCST particles, the days 1, 2, and 6 after the preparation, and the y-axis of FIG. 4B represents the absolute values of the fluorescence signals immediately after the UCST particles were produced, , 2 days and 6 days, respectively, and the x-axis in FIGS. 4A and 4B represents the cycle number.
  • FIG. 5 is a graph (red line, dark line) showing a result of performing a one-step reverse transcription quantitative PCR using UCST particles containing a reverse primer according to an embodiment of the present invention.
  • a solid line represents a positive control signal
  • a dotted line represents a false positive control signal by a primer duplex
  • a control signal no template control signal generated without a template reaction.
  • Cycle number, and the y-axis represents the standardized fluorescence intensity.
  • FIG. 6 is a graph showing the results of performing one step reverse transcription quantitative PCR using UCST particles including a reverse primer and a probe as one embodiment of the present invention.
  • the x-axis in the graph of Fig. 6 represents the cycle number and the y-axis represents the absolute value of the fluorescence signal.
  • FIG. 7 is a graph illustrating the effect of increasing the temperature of hydrogel microparticles containing UCST particles above UCST when the UCST material is melted and the fluorescence-labeled nucleic acid contained therein flows out of the hydrogel microparticles It is the figure which confirmed the state that it became.
  • FIG. 8 is a graph showing the relationship between the temperature of the UCST particles and the UCST particles when the temperature of the UCST particles is increased to UCST or higher.
  • FIG. 9 is an analysis of nucleic acid amplification reaction of hydrogel microparticles containing agarose as a UCST material according to an embodiment of the present invention.
  • FIG. 10 is an analysis of nucleic acid amplification reaction of hydrogel microparticles containing gelatin with UCST material according to an embodiment of the present invention.
  • FIG. 11 is an analysis of nucleic acid amplification reaction of hydrogel microparticles containing LMPA as a UCST material according to an embodiment of the present invention.
  • FIG. 12 is an analysis of nucleic acid amplification reaction of hydrogel microparticles containing PEG-aCD with UCST material as one embodiment of the present invention.
  • FIG. 13 shows the results of comparing the PCR efficiencies of one embodiment of the present invention (one primer immobilized with UCST) and Comparative Examples 2 and 3 (pair primer immobilized and one primer immobilized without UCST).
  • One embodiment of the present invention is a UCST polymer matrix having an Upper Critical Solution Temperature (UCST) at 20 to 90 < 0 >C;
  • UCST Upper Critical Solution Temperature
  • PCR polymerase chain reaction
  • the UCST particle may further include a reverse transcription (RT) primer to the UCST particle, or an omni-directional primer or a reverse primer that is the PCR primer may be used as a reverse primer without further comprising a reverse primer .
  • One embodiment of the present invention is a PCR-based hydrogel microparticle to which at least one primer of an forward primer and a reverse primer of a target nucleic acid or a probe of a target nucleic acid as a polymerase chain reaction primer is immobilized, and a UCST (Upper Critical Solution Temperature) particles, and one or more primers of the omnidirectional primer and the reverse primer, or a probe of the target nucleic acid may be contained in the UCST particle and fixed to the hydrogel fine particle.
  • the hydrogel microparticles may comprise two or more UCST particles, and each of the two or more UCST particles may each include at least one of an omnidirectional primer, a reverse primer and a probe of a target nucleic acid.
  • UCST Upper Critical Solution Temperature particle
  • the UCST particles may be particles having an Upper Critical Solution Temperature (UCST) at 20 to 90 ° C, and may be phase-transitioned from a liquid phase or a liquid phase to a solid phase at a solid phase at the high- Lt; / RTI >
  • the UCST particles may be particles comprised of materials that are melted during a denaturation step during the PCR process.
  • the UCST particles can be stably stored in the UCST particles until 6 days have elapsed after the UCST particles are prepared so as to contain the primer or the probe, , Figs. 4A and 4B).
  • RT-qPCR One-step reverse transcription-quantitative PCR
  • RT-qPCR is a one-step RT-qPCR method in which a reverse transcription reaction and a quantitative PCR are sequentially performed in a single reaction vessel and used for reverse transcription and quantitative PCR reaction
  • Primers used only in primers and quantitative PCR are mixed in a single reaction vessel from the beginning, and primers which should participate in quantitative PCR reactions are not specifically reacted to probes used in the reverse transcription reaction or primers or target nucleic acids (RNA) used in the reverse transcription reaction Thereby causing a problem.
  • a temperature lower than a critical temperature i.e., a denaturation step
  • the primer or probe contained in the UCST particles may not be leaked to the outside of the UCST particle and may contain the primer or the probe very stably until immediately before the PCR reaction so that the false positive signal due to the primer duplex is suppressed during the PCR, It was confirmed that nucleic acid amplification reaction, particularly one-step reverse transcription quantitative PCR can be performed with sensitivity and accuracy (Experimental Examples 2 and 3, Figures 5 and 6)
  • At least one primer or probe of the omnidirectional primer and the reverse primer is contained in the UCST particle and is fixed to the hydrogel microparticle, Since the primer or probe contained in the UCST particle does not flow out of the UCST particle prior to the denaturation step and may contain the primer or the probe very stably until immediately before the nucleic acid amplification reaction, It is possible to effectively prevent the nonspecific signal generated by the probe and the primer.
  • the UCST particles are melted and the primer or the probe contained in the UCST particle is released into the void of the hydrogel fine particle, whereby the hydrogel fine particle It is possible to perform a highly efficient nucleic acid amplification reaction (Experimental Examples 4 to 6, Figs. 9 to 13).
  • the UCST particles may be selected from the group consisting of agarose, gelatin, collagen, low melting point agarose (LMPA) and PEG-aCD (mixture of polyethylene glycol and alpha-cyclodextrin) And may include one or more species.
  • the material constituting the UCST particles is not limited to the above-exemplified materials, and various UCST materials may be selected according to room temperature stability and nucleic acid amplification reactivity.
  • the UCST particles include agarose having a critical dissolution temperature of 80 to 90 ° C, gelatin having a critical dissolution temperature of 40 to 50 ° C, collagen having a critical dissolution temperature of 30 to 40 ° C, and the like can do.
  • the UCST particles may be modified materials of natural materials and include LMPA having a critical dissolution temperature of 60 to 80 ⁇ .
  • the UCST particles may include PEG-aCD having a critical dissolution temperature of 20 to 90 ° C and having a UCST property due to molecular attraction.
  • the hydrogel microparticles may have a porous structure including pores.
  • the porosity of the microparticles is between 10% and 80% by volume, more specifically between 20% and 70% by volume relative to the total volume of microparticles. . If it is out of the above range, the porosity may be deteriorated or the structural stability of the fine particle may be unstable, which may be disadvantageous to the nucleic acid amplification reaction.
  • the target nucleic acid primer includes, but is not limited to, one or more nucleic acids of DNA, RNA, LNA, and PNA.
  • the target nucleic acid primer may be 10-100 base pairs (bp), more specifically 20-50 base pairs, but the sequence type and sequence length of the nucleic acid primer can be varied without limitation depending on the target nucleic acid.
  • the hydrogel microparticles may include one of the primer and the reverse primer in the inside of the UCST particle.
  • another primer not contained in the UCST particle May be fixed to the outside of the hydrogel fine particles or inside the pores of the hydrogel fine particles.
  • the hydrogel microparticles may contain a probe of the target nucleic acid in the UCST particle.
  • only one primer of the forward primer and the reverse primer may be included in the hydrogel It may be fixed within the pores of the fine particles, or both the omni-directional primer and the reverse primer may not be fixed within the pores of the hydrogel microparticles.
  • the primer that is not contained in the UCST particle contains a hydrogel microparticle-immobilized functional group at the 5 'end or the 3' end of the base sequence of the primer or cross-links with the hydrogel, Or may be fixed within the cavity. And may be immobilized by covalent bonding or peptide bonding with a hydrogel monomer within the pores of the hydrogel fine particles.
  • the functional group that can be linked to the hydrogel monomer through a covalent bond may include acrydite.
  • the functional group that can be connected to the hydrogel through a peptide bond includes an amine group, A carboxyl group and the like.
  • the primer modified with an acrylate functional group at the 5 'end of the nucleotide sequence may be immobilized on the hydrogel when the hydrogel monomer is converted into a polymer in the preparation of the hydrogel microparticles.
  • the hydrogel microparticles may have a particle size of, for example, an average particle size of 10 ⁇ m to 500 ⁇ m, and more specifically an average particle size of 100 ⁇ m to 300 ⁇ m.
  • the shape is not limited as long as it is a three-dimensional structure, and more specifically, spherical, hemispherical, disk-shaped, plate-shaped, and the like are exemplified.
  • the material of the hydrogel fine particles can be used without limitation as long as it is a pre-polymer that can be solidified. Specifically, polyethylene glycol-diacrylate (PEG-DA) or polyacrylamide (PA) May be used.
  • PCR fluorescent labeling can be performed by using a nonspecific fluorescent labeling method that fluoresces by interrupting the amplification products generated during the nucleic acid amplification reaction using SYBR green I dye or the like and by using a probe to identify the amplification product generated by the nucleic acid amplification reaction
  • a specific fluorescent labeling method which binds to a sequence and fluoresces by nucleic acid amplification reaction can be used.
  • the UCST particle may include a probe therein. More specifically, the probe may be an optional fluorescent probe.
  • the fluorescent probe binds to the target nucleic acid and provides a fluorescence signal to enable detection of the target nucleic acid in real time.
  • the target nucleic acid is amplified by polymerase chain reaction, and the fluorescence intensity is also increased. Therefore, the target nucleic acid to be amplified can be quantified by detecting fluorescence intensity.
  • the fluorescence probe can be used without limitation as long as it exhibits fluorescence by complementarily binding to a target nucleic acid.
  • the selective fluorescent probe may include a TaqMan probe or the like.
  • the TaqMan probe is a nucleic acid having a 5 'end modified with a fluorescent substance (FAM or the like) and a 3' end modified with a quencher substance (BHQ or the like) and subjected to an annealing step
  • FAM fluorescent substance
  • BHQ quencher substance
  • the 5 ' ⁇ 3' exonuclease possessed by the Taq DNA polymerase during the extension reaction is inhibited by the quencher on the probe, As a result, the fluorescent dye is released from the probe, so that the inhibition by the fiber is released and the fluorescence is expressed.
  • the UCST particle or the hydrogel microparticle may comprise at least one of an encoder for providing target nucleic acid primer or probe information and a fluorescent marker for providing quantitative information of the nucleic acid to be amplified, UCST particles or in the pores of the microparticles.
  • the encoder refers to a substance that distinguishes between nucleic acid primers or probes in each UCST particle or hydrogel microparticle in terms of color or shape.
  • the encoder may be a dye emitting fluorescence of various colors, a quantum dot, Metal, plastic, glass, silicon, or the like can be used.
  • the target nucleic acid primer in each UCST particle or hydrogel microparticle by changing the size or shape of the UCST particle or the hydrogel microparticle itself or by marking the surface of the UCST particle or hydrogel microparticle, Probes can be distinguished.
  • the target nucleic acid primer or probe in each UCST particle or hydrogel microparticle can be distinguished by specifying the position in the array of UCST particles or each hydrogel microparticle.
  • the present invention provides a method for producing UCST particles, wherein at least one primer of an omnidirectional primer and a reverse primer of a target nucleic acid or a probe of a target nucleic acid is added to a liquid UCST material and then the mixture is coagulated to prepare UCST particles
  • the method comprising the steps of:
  • the step of preparing the UCST particles may include a step of completely dissolving the UCST material in a liquid phase by heating the UCST material to a critical dissolution temperature or higher before adding and mixing at least one of an omnidirectional primer and a reverse primer of a target nucleic acid to a liquid UCST material, As shown in FIG.
  • coagulating the UCST material after mixing may include cooling the UCST material to below the critical dissolution temperature and solidifying the solid phase.
  • the present invention provides a method for producing the hydrogel microparticles, wherein one or more primers of an omnidirectional primer and a reverse primer of a target nucleic acid or a probe of a target nucleic acid are put into a liquid UCST material, ; Mixing the prepared UCST particles, a hydrogel monomer and a photoinitiator to prepare a prepolymer solution; And discharging the prepolymer solution in a droplet form and curing the prepolymer solution to prepare hydrogel microparticles.
  • the prepolymer solution may be prepared by mixing the primer and the reverse primer, which are not contained in the UCST particles, As shown in FIG.
  • the prepolymer solution may further include one of the forward primer and the reverse primer have.
  • pre-polymer means a prepolymer in which polymerization or polycondensation reaction is stopped at an appropriate stage in order to facilitate molding of the polymer.
  • the method may further include a cleaning step after the hydrogel microparticles are prepared, and the uncoupled UCST material, the void-derived polymer, and the like may be removed through the cleaning step.
  • the step of ejecting the prepolymer solution in droplet form includes a microchannel method, a piezo method or a solenoid value method, microspotting, and the like Thereby making it possible to produce fine particles of various shapes and sizes.
  • the UCST particle production method may be used to inject a target nucleic acid primer or a probe differently depending on the type of target nucleic acid, so that a plurality of UCST particles containing different target nucleic acid primers or probes Can be produced.
  • the UCST particle may further include at least one of an encoder that provides information of the target nucleic acid primer or probe included in each UCST particle, and a fluorescent marker that provides quantitative information of the target nucleic acid to be amplified.
  • the present invention also provides a method for producing a hydrogel microparticle, comprising the steps of: injecting a target nucleic acid primer or a probe differently according to the kind of a target nucleic acid, thereby preparing a plurality of target nucleic acid primers or probes Hydrogel microparticles can be prepared.
  • the prepolymer solution may further comprise at least one of an encoder providing information of the target nucleic acid primer or probe contained in each hydrogel microparticle and a fluorescent marker capable of providing quantitative information of the target nucleic acid to be amplified .
  • the step of preparing the prepolymer solution may further comprise a porosity inducing polymer in the solution.
  • the step of preparing the prepolymer solution may further include adjusting the size of the voids formed in the hydrogel microparticles by modifying the size of the void-derived polymer contained in the solution.
  • PEG polyethylene glycol
  • PAM polyacrylamide
  • PEG 200, PEG 300, PEG 400, PEG 600, PEG 1000, PEG 1500, PEG 2000, PEG 3000, PEG 3350, PEG 4000, PEG 6000, PEG 8000, PEG 10000, PEG 12000, PEG 20000, PEG 35000, PEG 40000 (manufacturer: Sigma Aldrich) have.
  • the curing of the hydrogel fine particles is performed by maintaining the shape of the hydrogel fine particles before curing, and if the shape can be maintained, the method such as optical, chemical, or thermal curing methods is not limited. As shown in Fig.
  • One embodiment of the present invention can provide a nucleic acid amplification apparatus including at least one UCST particle.
  • the nucleic acid amplification apparatus may include a plurality of UCST particles each including a primer or a probe for different target nucleic acids.
  • an embodiment of the present invention can provide a nucleic acid amplification apparatus including at least one hydrogel fine particle.
  • the nucleic acid amplification apparatus may include a plurality of hydrogel microparticles each including a primer or a probe for different target nucleic acids.
  • the apparatus may further comprise a reaction chamber, which may comprise an array or tube in which the UCST particles or hydrogel microparticles are arranged.
  • a reaction chamber which may comprise an array or tube in which the UCST particles or hydrogel microparticles are arranged.
  • the material of the array according to an exemplary embodiment is not limited to a material type as long as temperature conditions of nucleic acid amplification reaction such as glass, plastic, polymer, and silicon can be applied.
  • one embodiment of the present invention provides a method of forming a UCST particle, comprising: injecting one or more UCST particles into a reaction chamber; Injecting a solution containing one or more target nucleic acids into the reaction chamber; And amplifying the target nucleic acid by polymerase chain reaction (PCR) of the target nucleic acid.
  • PCR polymerase chain reaction
  • the step of amplifying the target nucleic acid may include the step of melting the UCST particles in the denaturation step during the PCR and allowing the primer or probe contained therein to flow out into the chamber.
  • the step of amplifying the target nucleic acid as an embodiment may include a step in which the UCST particles are melted in the denaturation step in the PCR so that the primer or the probe contained therein flows out of the UCST particle.
  • one embodiment of the present invention provides a method of preparing a hydrogel microparticle, comprising: injecting at least one hydrogel microparticle into a reaction chamber; Injecting a solution containing one or more target nucleic acids into the chamber; And amplifying the target nucleic acid by polymerase chain reaction (PCR) of the target nucleic acid.
  • PCR polymerase chain reaction
  • the step of amplifying the target nucleic acid may include the step of melting the UCST particles in the denaturation step during the PCR so that primers or probes contained in the UCST particles are released into the voids of the hydrogel microparticles.
  • the one or more UCST particles may each comprise at least one of an omnidirectional primer, a reverse primer and a probe for the same target nucleic acid, or may each comprise a primer or probe for a different target nucleic acid.
  • the hydrogel microparticles may comprise two or more UCST particles, and the two or more UCST particles may each include one or more of an omni-directional primer, a reverse primer, and a probe for the same target nucleic acid.
  • the at least one hydrogel microparticle may comprise a primer or probe for different target nucleic acids, respectively.
  • the solution containing the target nucleic acid may further include a primer containing LNA (Locked Nucleic Acid) such as a 3'-locked nucleic acid primer, Taq polymerase, and the like.
  • the reaction chamber may include an array or a tube in which the UCST particles or the hydrogel microparticles are arranged.
  • the method may further comprise analyzing nucleic acids that are polymerized within the one or more respective UCST particles. In one embodiment, the method may further comprise analyzing the nucleic acid polymerized within the one or more respective hydrogel microparticles. In another embodiment, the method further comprises quantitatively analyzing in real time quantitative analysis of the nucleic acid polymerized in the at least one respective UCST particle or hydrogel microparticle that is co-polymerized with the step of performing the polymerase chain reaction, As such, different types of target nucleic acids can be simultaneously amplified and detected in real time and quantitatively analyzed.
  • UCST particles containing a reverse primer were prepared using LMPA as a UCST material.
  • the LMPA was completely dissolved in a liquid state at a temperature above the critical melting temperature, and then the reverse primer solution of the target nucleic acid was added to the liquid UCST material at a rate of 10% v / v based on the total volume of the liquid UCST material. To cool down and solidify to produce solid UCST particles.
  • sequence of the target nucleic acid and the reverse primer used here is as follows.
  • Target nucleic acid 5'-AGGGCATTTTGGACAAAGCGTCTACGCTGCAGTCCTCGCTCACTGGGCACGGTGAGCGTGAACACAAACCCCAAAATCCCCTTAGTCAGAGGTGACAGGATTGGTC-3 '(SEQ ID NO: 1)
  • UCST particles (Example 2) including a reverse primer and a probe were prepared using LMPA as a UCST material.
  • solid UCST particles containing the reverse primer of SEQ ID NO: 2 and the probe of SEQ ID NO: 4 for the target nucleic acid of SEQ ID NO: 1 were prepared in the same manner as in Preparation Example 1,
  • hydrogel microparticles containing UCST particles of Examples 3-6 were prepared using four UCST materials of agarose, gelatin, LMPA and PEG-aCD, respectively.
  • sequence of the target nucleic acid and the forward primers and reverse primers used are as follows, and the probe was used for TaqMan TM probe.
  • the omni-directional primer was immobilized in hydrogel microparticles using an acrylate.
  • Target nucleic acid 5'-CCTGGCACCCAGCACAATGAAGATCAAGATCATTGCTCCTCCTGAGCGCAAGTACTCCGTGTGGATCGGC-3 '(SEQ ID NO: 5)
  • the UCST material is completely dissolved in a liquid state at the respective critical melting temperature or higher, and then a reverse primer solution is added to the liquid UCST material at a rate of 10% v / v based on the total volume of the liquid UCST material. To cool down and solidify to produce solid UCST particles.
  • the prepared solution was discharged in a droplet form and cured by UV exposure (360 nm wavelength, 35 mJ / cm 2 ) for 1 minute to prepare hydrogel fine particles having an average particle diameter of 400 ⁇ m.
  • the microparticles were washed with PBS 1X buffer to remove uncured materials.
  • One-step reverse transcription-quantitative PCR was performed as follows to confirm whether the UCST particles according to one embodiment of the present invention had a primer or probe storage stability .
  • the UCST particles of Example 1 prepared in [Preparation Example 1] were injected into a channel filled with PBS 1X buffer and stored at a temperature of 40 ° C. Immediately after the preparation, 1 day after the preparation, 2 days after the preparation, 6 days after the production, the UCST particles and the omni-directional primer of SEQ ID NO: 3 were injected into the channel to perform one step reverse transcription quantitative PCR.
  • One-step reverse transcription-PCR was carried out under conditions of reverse transcription at 42 ° C for 10 min, denaturation at 95 ° C for 4 sec for quantitative PCR, and DNA synthesis at 55 ° C for 30 sec for quantitative PCR.
  • the denaturation step and the DNA synthesis step of the quantitative PCR process were repeated 40 times.
  • the fluorescence intensity of the particles was observed 40 times for 1 second immediately after the DNA synthesis step of the quantitative PCR process, and the fluorescence intensity was measured, and the results are shown in FIGS. 4A and 4B.
  • RNA target nucleic acid
  • One-step reverse transcription-quantitative PCR was performed in a nucleic acid amplification reaction using UCST particles containing UCST particles containing a reverse primer as one embodiment of the present invention. The same procedure as in Experimental Example 1 was carried out.
  • the UCST particles used were the UCST particles of Example 1 prepared in [Preparation Example 1] above.
  • the reaction solution containing the reverse primer and the omnidirectional primer was inserted into the channel without using UCST particles as Comparative Example 1, and one step reverse quantitative PCR was performed in the same manner as in Experimental Example 1.
  • the results of the one-step reverse transcription quantitative PCR of Example 1 and Comparative Example 1 are shown in Fig.
  • the red line (dark line) in FIG. 5 shows the results of the one-step reverse transcription quantitative PCR of Comparative Example 1
  • the blue line (soft line) shows the one-step reverse transcription quantitative PCR results using the UCST particles of the present invention, (positive control signal)
  • the dotted line represents a control signal (no template control signal) generated without reaction of the template, which is a false positive control signal by the primer duplex.
  • the one-step reverse transcription quantitative PCR was performed only in the reverse reaction and the quantitative PCR in one reaction vessel, and was used only in the primer (forward primer in this Experimental Example 2) and the quantitative PCR which are both used for reverse transcription and quantitative PCR reaction Since the primer (reverse primer in Experimental Example 2) is mixed in one reaction vessel from the beginning, the reverse primer, which should participate only in the quantitative PCR reaction, acts nonspecifically on the reverse primer or the target nucleic acid (RNA) ≪ / RTI >
  • Example 1 when comparing the dotted lines of Comparative Example 1 and Example 1 of FIG. 5, the Ct value of Example 1 is delayed by 6 or more. This shows that the sensitivity is improved by about 100 times or more, , It can be seen that the false positive signal due to the primer duplex is suppressed when the one step reverse transcription quantitative PCR is performed and the nucleic acid amplification reaction can be performed with higher sensitivity and accuracy.
  • one-step RT-qPCR was performed as follows in the nucleic acid amplification reaction using UCST particles containing a reverse primer and a probe.
  • UCST particles of Example 2 including a reverse primer and a probe were used in place of the UCST particles of Example 1.
  • Example 4 prepared in [Preparation Example 3] were injected into a channel filled with PBS 1X buffer, and the fluorescence of the particles was observed for 100 seconds at a temperature of 80 ° C.
  • the decrease in the fluorescence of the particles means that the primer contained in the UCST particles is discharged out of the hydrogel fine particles.
  • Fig. 8 shows the result of repeating the same experiment three times independently using the same fine particles.
  • the UCST is melted at a temperature condition of 40 to 50 ° C., which is a temperature condition of the denaturation step, and the primer contained in the UCST particle is released to participate in the amplification reaction freely before and after the UCST temperature .
  • nucleic acid amplification reaction was performed using hydrogel microparticles containing various UCST particles.
  • Each of the hydrogel microparticles prepared in [Manufacturing Example 3] was placed in a channel, and a sample (nucleic acid template) to be analyzed was transferred to a PCR master mix (reagent containing enzyme, buffer, dNTP, Mg2 + ), Put into a channel, and subjected to PCR.
  • a sample nucleic acid template
  • PCR master mix reagent containing enzyme, buffer, dNTP, Mg2 +
  • the one-primer immobilized with UCST of Example 4 prepared in [Preparation Example 3] was used.
  • Example 3 the same hydrogel particles (Pair primer immobilized: Comparative Example 2) as in Example 4 were prepared except that the UCST particles were not included and the forward and reverse primers were all fixed inside the voids of the hydrogel fine particles.
  • One primer immobilized without (UCST) particle was prepared in the same manner as in Example 4, except that the omnidirectional primer was not contained in the UCST particles but was immobilized in the hydrogel fine particle voids and UCST particles not containing the reverse primer and the probe were included.
  • UCST Comparative Example 3 was used.
  • FIG. 13 shows the results of the PCR.
  • the fluorescence intensity was very low irrespective of whether the unidirectional primer was immobilized in the hydrogel fine particles, ,
  • an embodiment of the present invention shows a very high PCR efficiency. It is considered that the present invention prevents formation of primer duplex due to non-specific binding between the primer or the primer and the probe due to the use of the UCST particle and that the primer contained in the UCST particle has a high degree of freedom during the PCR process .

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Abstract

Dans la présente invention, lors de l'amplification d'un acide nucléique par incorporation d'au moins une amorce parmi une amorce sens et une amorce antisens et/ou d'une sonde dans une particule à température de solution critique supérieure (UCST) ou lors de l'amplification d'un acide nucléique par incorporation d'au moins une amorce parmi l'amorce sens et l'amorce antisens et/ou d'une sonde dans une particule UCST et par fixation à une particule fine d'hydrogel, l'amorce ou la sonde contenue dans la particule UCST pouvant être déchargée dans une certaine plage de température. Par conséquent, la formation de dimères d'amorce peut être empêchée tout en obtenant également une excellente efficacité d'amplification PCR.
PCT/KR2018/007523 2017-07-04 2018-07-03 Particule avec un matériau ucst appliqué à celle-ci, et procédé d'amplification d'acide nucléique l'utilisant Ceased WO2019009592A2 (fr)

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US16/628,346 US20200239944A1 (en) 2017-07-04 2018-07-03 Particle with ucst material applied thereto, and nucleic acid amplification method using same
CN201880057430.3A CN111356771A (zh) 2017-07-04 2018-07-03 适用ucst材料的粒子及利用其的核酸扩增方法

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KR10-2017-0084595 2017-07-04
KR20170084595 2017-07-04
KR10-2018-0076483 2018-07-02
KR1020180076483A KR102088877B1 (ko) 2017-07-04 2018-07-02 Ucst 소재를 적용한 입자 및 이를 이용한 핵산 증폭 방법

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US6642000B1 (en) * 1999-11-12 2003-11-04 University Of Chicago PCR amplification on microarrays of gel immobilized oligonucleotides
WO2008146649A1 (fr) * 2007-05-25 2008-12-04 Olympus Corporation Gel contenant une enzyme, et coffret d'amplification d'acide nucléique
KR101670232B1 (ko) * 2013-10-28 2016-10-31 한국과학기술연구원 다공성 구조체 및 이의 제조방법
KR101746055B1 (ko) * 2016-05-03 2017-06-12 한국과학기술연구원 코어-쉘 구조의 다공성 구조체 및 이를 이용한 핵산 증폭 방법

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