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WO2016148090A1 - Gene examination method using stimulation-responsive magnetic nanoparticles - Google Patents

Gene examination method using stimulation-responsive magnetic nanoparticles Download PDF

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Publication number
WO2016148090A1
WO2016148090A1 PCT/JP2016/057870 JP2016057870W WO2016148090A1 WO 2016148090 A1 WO2016148090 A1 WO 2016148090A1 JP 2016057870 W JP2016057870 W JP 2016057870W WO 2016148090 A1 WO2016148090 A1 WO 2016148090A1
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Prior art keywords
nucleic acid
conjugate
acid amplification
responsive
stimulus
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PCT/JP2016/057870
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French (fr)
Japanese (ja)
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貴寛 関川
小毛 謝
大西　徳幸
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静岡県公立大学法人
Jnc株式会社
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Publication of WO2016148090A1 publication Critical patent/WO2016148090A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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

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  • the present invention relates to a genetic testing method using stimulus-responsive magnetic nanoparticles.
  • Analyzing and identifying genes by purifying nucleic acids from samples containing nucleic acids is frequently performed in the fields of hygiene tests, clinical tests, and research.
  • Cryptosporidium a protozoan that parasitizes the digestive tract of various animals, exhibit strong chlorine tolerance, often causing mass diarrhea via water. Therefore, Cryptosporidium has become the most prominent protozoa currently in clinical or public health terms.
  • Cryptosporidium The detection of Cryptosporidium is usually performed by visual observation under a microscope.
  • this method requires a lot of labor for detection because of complicated operation, low speed, and low detection rate. Therefore, in recent years, genetic techniques have also been used for the study of Cryptosporidium, and it has become relatively easy to identify and type species.
  • genes of various organisms are being elucidated by the development of genetic engineering, and it is important to elucidate the functions and biological roles of these elucidated genes. Furthermore, in such functional analysis of genes, it is extremely important to know what genes are expressed in each tissue of an organism. For example, as one technique for analyzing gene expression in such a tissue, mRNA is collected from the tissue and the mRNA is analyzed.
  • the magnetic bead method has been used as a method for separating nucleic acids from the environment and biological samples (Patent Document 1, Non-Patent Documents 1 and 2).
  • Patent Document 1 Non-Patent Documents 1 and 2
  • the magnetic fine particles used in the magnetic bead method magnetic fine particles (for example, Dynabeads (registered trademark)) whose particle size is designed to be micro size are used in order to give the magnetic beads magnetic separation ability. .
  • nucleic acid DNA / RNA
  • the nucleic acid and magnetic fine particles are separated before performing nucleic acid amplification. It was necessary to purify the nucleic acid, and the work was complicated.
  • an object of the present invention is to provide a novel genetic test method capable of performing reverse transcription reaction and / or nucleic acid amplification as it is without requiring complicated steps such as subsequent nucleic acid separation or nucleic acid purification.
  • the present inventors collect nucleic acids using stimulus-responsive magnetic nanoparticles to which an affinity substance for a target nucleic acid is bound, and perform nucleic acid amplification in the presence of a nonionic surfactant, thereby stimulating It has been found that it can be subjected to a reverse transcription reaction and / or a nucleic acid amplification test in a state in which responsive magnetic nanoparticles are contained, and the present invention has been completed.
  • the present invention has the following configuration.
  • a genetic test method for recovering nucleic acid in a test sample and performing nucleic acid amplification comprising the following steps (1) to (4): (1) A step of generating a conjugate of the nucleic acid and the adsorbent having an affinity substance with respect to the target nucleic acid and the nucleic acid in an aqueous solution containing the test sample (2) the stimulus responsive magnetic nanoparticle A step of aggregating the conjugate (3) a step of recovering the aggregated conjugate with a magnet (4) a nucleic acid amplification of the recovered conjugate in the presence of a nonionic surfactant Step 2 to perform 2.
  • the target nucleic acid (DNA / RNA) can be easily recovered regardless of the type of the test sample and without using a carefully handled reagent. Furthermore, the reverse transcription reaction and / or nucleic acid amplification of the obtained nucleic acid can be carried out as it is without requiring complicated steps such as subsequent nucleic acid separation or nucleic acid purification.
  • FIG. 1 is a schematic configuration diagram of an adsorbent in a method according to an embodiment of the present invention.
  • FIG. 2 is a graph showing the effect of reducing the inhibition of PCR by the concentration of TM-AC by adding a nonionic surfactant.
  • the present invention is a genetic test method for recovering nucleic acid in a test sample and performing nucleic acid amplification, and includes the following steps (1) to (4).
  • a step of generating a conjugate of the nucleic acid and the adsorbent having an affinity substance with respect to the target nucleic acid and the nucleic acid in an aqueous solution containing the test sample (2) the stimulus responsive magnetic nanoparticle
  • a step of aggregating the conjugate (3) a step of recovering the aggregated conjugate with a magnet (4) a nucleic acid amplification of the recovered conjugate in the presence of a nonionic surfactant Process to perform
  • the genetic testing method of the present invention is to perform genetic testing by collecting nucleic acid from a test sample and amplifying the nucleic acid.
  • first there is a step of recovering a nucleic acid from a test sample.
  • This step is a step of generating an adsorbent / nucleic acid conjugate as the first stage of nucleic acid recovery.
  • Test sample The test sample is not particularly limited, and for example, organisms such as animals (including humans), plants, microorganisms (including protozoa and protozoa) can be targeted. Moreover, as a test sample, for example, even a protozoan that forms oocysts such as Cryptosporidium can be applied as the test sample of the present invention. Other test samples include viruses, bacteria, fungi, and the like.
  • nucleic acid is used as a concept including DNA, RNA, and derivatives thereof.
  • nucleic acids from animal-derived test samples include blood free DNA from blood components such as serum and plasma, DNA such as viral DNA, blood free messenger RNA, RNA such as viral RNA, protozoa and bacteria-derived DNA, ribosomal RNA, messenger RNA and the like.
  • the adsorbent can be used for recovering nucleic acids.
  • the adsorbent has an affinity substance for stimulus-responsive magnetic nanoparticles and nucleic acids, and is formed by binding both.
  • stimulus-responsive magnetic nanoparticles are used for recovery of nucleic acids in an aqueous solution containing a test sample.
  • stimulation-responsive magnetic nanoparticles it is not necessary to purify nucleic acids by separating nucleic acids from stimulation-responsive magnetic nanoparticles during nucleic acid amplification, which will be described later. It can be carried out.
  • the stimulus-responsive magnetic nanoparticles used in the present invention are obtained using a stimulus-responsive polymer and a magnetic substance (magnetic fine particles).
  • the stimulus-responsive polymer is a magnetic nanoparticle that undergoes a structural change in response to an external stimulus and can adjust aggregation and dispersion.
  • the type of stimulation is not particularly limited, and examples thereof include various physical or chemical signals such as temperature, light, acid, base, pH, or electricity.
  • a temperature-responsive polymer that can be aggregated and dispersed by temperature change can be used.
  • the temperature responsive polymer include a polymer having a lower critical solution temperature (hereinafter also referred to as LCST) and a polymer having an upper critical solution temperature (hereinafter also referred to as “UCST”).
  • Examples of the polymer having a lower critical solution temperature that can be used in the present invention include Nn-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, N- Acryloylpiperidine, N-acryloylmorpholine, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine, N-methacryloylmorpholine Polymers composed of N-substituted (meth) acrylamide derivatives such as: hydroxypropyl cellulose, polyvinyl alcohol partially acetylated product, polyvinyl methyl ether, (polyoxyethylene Polyoxypropylene) block copolymer, polyoxyethylene alkylamine derivatives such as poly
  • a copolymer of N-isopropylacrylamide and Nt-butylacrylamide can be preferably used.
  • Examples of the polymer having an upper critical solution temperature that can be used in the present invention include, for example, at least one monomer selected from the group consisting of acryloylglycinamide, acryloylnipecotamide, acryloylasparaginamide, acryloylglutamineamide, and the like.
  • the polymer which becomes is mentioned.
  • the copolymer which consists of these at least 2 types of monomers may be sufficient.
  • These polymers include other copolymers such as acrylamide, acetylacrylamide, biotinol acrylate, N-biotinyl-N'-methacryloyl trimethylene amide, acroyl sarcosine amide, methacryl sarcosine amide, acroyl methyl uracil, etc.
  • Possible monomers may be copolymerized in a range having an upper critical solution temperature.
  • a pH-responsive polymer that can be aggregated and dispersed by pH change can be used as the stimulus-responsive polymer.
  • examples of such a pH-responsive polymer include a polymer containing a group such as carboxyl, phosphoric acid, sulfonyl, and amino as a functional group.
  • acrylic acid methacrylic acid, maleic acid, vinyl sulfonic acid, vinyl benzene sulfonic acid, phosphoryl ethyl (meth) acrylate, aminoethyl methacrylate, aminopropyl (meth) acrylamide, dimethylaminopropyl (meth)
  • examples include polymers containing acrylamide or a salt thereof as a copolymerization component.
  • Magnetic fine particles which are magnetic substances, are composed of, for example, polyhydric alcohol and magnetite.
  • the polyhydric alcohol is not particularly limited as long as it is an alcohol structure having at least two hydroxyl groups in a structural unit and capable of binding to iron ions.
  • Examples include dextran, polyvinyl alcohol, mannitol, sorbitol and cyclodextrin.
  • Japanese Patent Application Laid-Open No. 2005-82538 discloses a method for producing a particulate magnetic material using dextran.
  • the compound which has an epoxy like a glycidyl methacrylate polymer and forms a polyhydric alcohol structure after ring-opening can also be used.
  • the fine particle magnetic material (magnetic fine particles) prepared using such a polyhydric alcohol preferably has an average particle size of 0.9 nm or more and less than 1000 nm so as to have good dispersibility.
  • the average particle size is preferably 2.9 nm or more and less than 200 nm, in particular, in order to increase the detection sensitivity of the target detection target.
  • the dispersibility in a liquid becomes favorable because the average particle diameter of a magnetic fine particle is the said range.
  • the affinity substance used in the present invention is not particularly limited as long as it binds to a nucleic acid.
  • nucleic acids, aptamers, substances having a cationic functional group for example, substances having a primary amino group, secondary amino group, tertiary amino group, quaternary ammonium group or guanidino group
  • substances having an anionic functional group for example, substances having a primary amino group, secondary amino group, tertiary amino group, quaternary ammonium group or guanidino group
  • proteins that interact with the target substance eg, enzymes, receptors
  • chelating agents eg., enzymes, receptors, chelating agents, and the like.
  • the binding method between the stimulus-responsive magnetic nanoparticles and the affinity substance is not particularly limited.
  • substances having affinity for example, biotin and streptavidin or avidin
  • a stimulus-responsive magnetic nanoparticle is bound to an affinity substance.
  • biotin is bonded to the stimulus-responsive polymer by adding biotin or the like to a polymerizable functional group such as methacryl or acryl as described in International Publication No. 2001/009141.
  • a monomer is used and copolymerized with other monomers to immobilize streptavidin.
  • labeling of the affinity substance with biotin or the like is performed according to a conventional method. When the affinity substance labeled with biotin and the streptavidin-immobilized stimulus-responsive polymer are mixed, the stimulus-responsive polymer and the affinity substance are bound via the binding between biotin and streptavidin.
  • the stimulus-responsive magnetic nanoparticle 1 includes a particulate magnetic substance 2, and a stimulus-responsive polymer 3 is bonded to the surface of the magnetic substance 2.
  • Streptavidin 4 is bound to the stimulus-responsive polymer 3, and an affinity substance 6 for the detection target 7 is bound via biotin 5.
  • a material in which a stimulus-responsive polymer and a particulate magnetic substance are combined is referred to as a stimulus-responsive magnetic nanoparticle, and when the stimulus is heat, it is referred to as a thermoresponsive magnetic nanoparticle. Yes.
  • the fine magnetic substance can be bonded to the stimulus-responsive polymer via a reactive functional group, or a polymerizable unsaturated bond is introduced into the active hydrogen or polyhydric alcohol on the polyhydric alcohol in the magnetic substance.
  • the graft polymerization may be performed by a method known in the art such as graft polymerization (for example, ADV.Polym.Sci., Vol.4, p111, 1965, J. Polymer Sci., Part-A, 3, p1031, 1965).
  • the adsorbent / nucleic acid conjugate described above is obtained by mixing the adsorbent and nucleic acid in an aqueous solution by any method, whereby the affinity substance in the adsorbent and the nucleic acid are combined, and the adsorbent / nucleic acid conjugate is formed. Generate.
  • the binding between the affinity substance and the nucleic acid means specific binding or adsorption, and is not necessarily a covalent binding, and may be binding utilizing ionic or biochemical affinity (affinity).
  • the mixing operation is not particularly limited as long as the adsorbent and the nucleic acid can come into contact with each other in an appropriate buffer. For example, it is sufficient to lightly invert or shake the test sample containing nucleic acid and the tube provided with the adsorbent, and examples thereof include an operation of mixing using a commercially available vortex mixer or the like.
  • step (2) A step of aggregating the conjugate under conditions where the stimuli-responsive magnetic nanoparticles are agglomerated
  • This step is a second step of nucleic acid recovery, and a conjugate of nucleic acid and adsorbent was obtained in step (1). Then, it is the process which puts on the conditions which a stimulus responsive polymer aggregates, and aggregates a conjugate
  • the container containing the mixture may be transferred to a thermostatic bath at a temperature at which the thermoresponsive polymer aggregates.
  • thermoresponsive polymers polymers having an upper critical solution temperature and polymers having a lower critical solution temperature.
  • thermoresponsive polymer when a polymer having a lower critical solution temperature with an LCST of 37 ° C. is used, the thermoresponsive polymer can be agglomerated by moving the container containing the mixture to a constant temperature bath at 37 ° C. or higher.
  • thermoresponsive polymer when a polymer having an upper critical solution temperature with a UCST of 5 ° C. is used, the thermoresponsive polymer can be agglomerated by transferring the container containing the mixture to a thermostatic bath of less than 5 ° C.
  • the lower critical solution temperature and the upper critical solution temperature are determined as follows, for example. First, a sample is put into a cell of an absorptiometer and the sample is heated at a rate of 1 ° C./min. During this time, the change in transmittance at 550 nm is recorded. Here, when the transmittance when the polymer is transparently dissolved is 100% and when the transmittance when the polymer is completely aggregated is 0%, the temperature at which the transmittance is 50% is obtained as LCST. In the case of UCST, the sample is cooled at a rate of 1 ° C./min, and thereafter obtained by the same method as LCST.
  • an acid solution or an alkali solution may be added to a container containing the mixture.
  • an acid solution or an alkaline solution is added to a container containing a dispersed mixture outside the pH range where the pH-responsive polymer undergoes a structural change, and the pH-responsive polymer undergoes a structural change in the container. What is necessary is just to change to the pH range to raise.
  • an acid solution may be added to a container containing a mixture dispersed at a pH of more than 5 so that the pH is 5 or less.
  • an alkaline solution may be added to a container containing a mixture dispersed at a pH lower than 10 so that the pH becomes 10 or higher.
  • the pH at which the pH-responsive polymer undergoes a structural change is not particularly limited, but is preferably pH 4 to 10, and more preferably pH 5 to 9.
  • a photoresponsive polymer when a photoresponsive polymer is used, light having a wavelength capable of aggregating the polymer may be irradiated to a container containing the mixture.
  • the preferred light for aggregation varies depending on the type and structure of the photoresponsive functional group contained in the photoresponsive polymer, but in general, ultraviolet light or visible light having a wavelength of 190 to 800 nm can be suitably used.
  • the strength is preferably 0.1 to 1000 mW / cm 2 .
  • the photoresponsive polymer is preferably one that is less likely to cause dispersion when irradiated with light used for turbidity measurement, in other words, that it can aggregate in that it can improve measurement accuracy.
  • the measurement accuracy can be improved by shortening the irradiation time.
  • Step of recovering aggregated conjugate with magnet This step is a step of recovering the detection target by separating the aggregate aggregated in step (2) with a magnet as the final stage of detection target recovery. By applying a magnetic force to the conjugate containing magnetic particles, the aggregated conjugate is separated from the mixed solution and recovered. As a result, the conjugate is separated from the contaminants containing the non-aggregated magnetic substance, and the detection target can be recovered.
  • the adsorbent when binding of nucleic acid and adsorbent is performed in a suitable tube, the adsorbent is held near the side wall of the tube by bringing the magnet close to the side wall of the tube from the outside, and the liquid that becomes the supernatant part from the inside of the tube By discharging the adsorbent, the adsorbent to which the nucleic acid is bound can be separated.
  • the magnetic force of a magnet or the like used for separating the adsorbent to which nucleic acid is bound varies depending on the magnitude of the magnetic force of the magnetic substance used.
  • a magnetic force capable of collecting the target magnetic substance can be appropriately used.
  • the material of the magnet for example, a material made of the above-described magnetic material can be used.
  • a neodymium magnet manufactured by 26 Co., Ltd.
  • the magnetic force of the neodymium magnet is preferably 3800 gauss or more.
  • Step of performing nucleic acid amplification on the recovered conjugate in the presence of a nonionic surfactant This step is a step of performing nucleic acid amplification using the nucleic acid in the conjugate recovered in the above step as a sample.
  • the stimulus-responsive magnetic nanoparticles in the adsorbent can inhibit reverse transcription reaction and nucleic acid amplification when the concentration of the stimulus-responsive magnetic nanoparticles in the reaction solution is high. For this reason, it has been necessary to purify the nucleic acid by separating the nucleic acid and the magnetic fine particles before the reverse transcription reaction or the nucleic acid amplification test.
  • by performing nucleic acid amplification in the presence of a nonionic surfactant it is possible to reduce inhibition of reverse transcription reaction and nucleic acid amplification by stimulus-responsive magnetic nanoparticles.
  • nucleic acid amplification in order to improve the detection sensitivity of the detection target, it is possible to perform nucleic acid amplification using the nucleic acid collected in the above step as it is as a sample.
  • the nucleic acid is RNA
  • a reverse transcription reaction is performed to obtain DNA, and then a nucleic acid amplification test is performed.
  • any conventionally known method can be employed using the RNA obtained in the above steps (1) to (3) as a template.
  • any method can be used as a method for nucleic acid amplification, such as polymerase chain reaction (PCR method), LAMP method, strand displacement amplification, reverse transcriptase strand displacement amplification, reverse transcriptase polymerase chain reaction, Examples include reverse transcription LAMP method, nucleic acid sequence-based amplification, transcription-mediated amplification and rolling circle amplification method, SmartAmp, and method reverse transcription SmartAmp method.
  • PCR method polymerase chain reaction
  • LAMP method strand displacement amplification
  • reverse transcriptase strand displacement amplification reverse transcriptase polymerase chain reaction
  • reverse transcriptase polymerase chain reaction examples include reverse transcription LAMP method, nucleic acid sequence-based amplification, transcription-mediated amplification and rolling circle amplification method, SmartAmp, and method reverse transcription SmartAmp method.
  • DNA amplification is generally performed by PCR.
  • any conventionally known PCR method can be employed using the DNA obtained in the above step or the DNA obtained by reverse transcription reaction of RNA as a template.
  • the nucleic acid amplification enzyme at the time of PCR amplification is any of Taq polymerase, Bst polymerase, or Aac polymerase, it can be suitably used.
  • Nonionic surfactant In this step, by performing nucleic acid amplification in the presence of a nonionic surfactant, it is possible to reduce inhibition of reverse transcription reaction and nucleic acid amplification by stimuli-responsive magnetic nanoparticles.
  • the nucleic acid amplification in the presence of a nonionic surfactant can reduce the inhibition of reverse transcription reaction and nucleic acid amplification by stimuli-responsive magnetic nanoparticles
  • the stimulus-responsive magnetic nanoparticle is not clear. It is presumed that the nonionic surfactant acts on the polymer on the particle surface to form micelles.
  • nonionic surfactant examples include polyoxyethylene derivatives.
  • examples of the polyoxyethylene derivative include polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene (10) octylphenyl ether (Triton X-100), and the like.
  • polyoxyethylene sorbitan monolaurate and polyoxyethylene (10) octylphenyl ether (Triton X-100) are preferable from the viewpoint of low viscosity and easy handling.
  • the concentration of the nonionic surfactant is preferably diluted to 0.1 to 5% by mass, more preferably 0.5 to 5% by mass. .
  • concentration of the nonionic surfactant is preferably diluted to 0.1 to 5% by mass, more preferably 0.5 to 5% by mass.
  • the stimulus-responsive nanoparticles remaining in the nucleic acid sample are preferably 0.1 to 1% by mass, more preferably 0.1 to 0.5% by mass It is. By being in the above range, inhibition of nucleic acid amplification by the stimulus-responsive magnetic nanoparticles can be reduced.
  • Example 1 Production of magnetic nanoparticles for recovery of oocysts (TM-AC) 1 mL of thermoresponsive magnetic nanoparticles to which avidin is bonded (Therma-Max LA Avidin, 4 g / L, JNC Corporation) and C.I. 20 ⁇ L of biotinylated monoclonal antibody specific for parvum oocysts (Crypt-a-Glo biotin reagent kit (20-fold concentrated), Waterborne) was combined and bound to magnetic nanoparticles for recovery of oocysts (Therma-Max-Anti-Cryptosporidium) : TM-AC).
  • PCR is a primer set specific for Cryptosporidium 18S rDNA gene forward: 5′-GGAAGGGTTGTATTATTATAGATAAAGAGAACC-3 ′ (SEQ ID NO: 2), reverse: 5′-CTCCCTCTCCGGGAATCGAA-3 (SEQ ID NO: 3) (each 5 ⁇ M) 0.8 ⁇ L and TaqMan Probe: TCTGACCZATCAGCTT (SEQ ID NO: 4) (1 ⁇ M) 2.0 ⁇ L, PCR reagent Premix EX Taq (Takara Bio Inc.) 10 ⁇ L, Tween 20 (Tween 20 (registered trademark), Molecular Biology Grade, manufactured by Promega), real-time PCR (LightCycC) Roche Diagnostics Inc.).
  • Tween 20 was adjusted to a final concentration of 5%.
  • the added amount of the positive control (5 ⁇ 10 6 copies / mL) was 2 ⁇ L, and the PCR reaction solution volume was 20 ⁇ L.
  • PCR conditions were “Hold; 1 cycle (95 ° C., 30 s), 2 Step PCR; 45 cycles (60 ° C., 30 s, 95 ° C., 10 s)”.
  • the Ct value (Threshold Cycle) was analyzed with LightCycler Nano software.
  • Comparative Example 1 Comparative Example 1 was tested in the same manner as in Example 1 except that Tween 20 was not added during PCR.
  • Example 2 Comparative Example 2] Example 2 was tested in the same manner as Example 1 except that the final concentration of TM-AC was adjusted to 0.5%. Comparative Example 2 was tested in the same manner as in Example 1 except that the final TM-AC concentration was adjusted to 0.5% and Tween 20 was not added during PCR.
  • the results of evaluating PCR inhibition by TM-AC are shown in FIG. 1 (white squares: no Tween 20 added).
  • the Ct value on the vertical axis represents the number of cycles when the PCR amplification product reaches a certain amount. The greater the amount of PCR amplification product, the smaller the Ct value, and it is not detected when it is not amplified.
  • the Ct value of the TM-AC addition concentration of 0.1% in Comparative Example 1 was almost the same as that of Control Example 1 (TM-AC addition 0%), which indicates that PCR was not inhibited. However, with the TM-AC addition of 0.5% in Comparative Example 2, the amplification rate was significantly delayed and the Ct value was 25.2. From this result, it can be seen that PCR is inhibited when 0.1 ⁇ L of TM-AC is added to the PCR tube (20 ⁇ L of reaction solution) (TM-AC final concentration 0.5%).
  • the present invention is not limited to the above-described embodiment, but includes modifications and improvements as long as the object of the present invention can be achieved.
  • the genetic testing method of the present invention it is possible to quickly and easily detect target microorganisms, particularly Cryptosporidium, from environmental / biological samples, and the obtained nucleic acid sample is subjected to complicated procedures such as subsequent nucleic acid purification. This is useful in clinical examinations and public health examinations, for example, because it does not adversely affect the reverse transcription reaction and / or nucleic acid amplification test as it is without requiring any additional steps.
  • target microorganisms particularly Cryptosporidium

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Abstract

The present invention addresses the problem of providing a method for easily examining genes regardless of the classification of a test sample and without using a reagent that requires cautious handling. The present invention relates to a gene examination method wherein nucleic acid is collected from a test sample and nucleic acid amplification is performed. The gene examination method includes steps (1)-(4). (1) A step wherein stimulation-responsive magnetic nanoparticles and a conjugate of a target nucleic acid and of an adsorption material that includes a substance that has affinity for said nucleic acid are generated in an aqueous solution that includes a test sample, (2) a step wherein the conjugate is agglomerated under conditions under which the stimulation-responsive magnetic nanoparticles agglomerate, (3) a step wherein the agglomerated conjugate is collected by means of a magnet, and (4) a step wherein nucleic acid amplification is performed on the collected conjugate in the presence of a non-ionic surfactant.

Description

刺激応答性磁性ナノ粒子を用いた遺伝子検査方法Genetic testing method using stimulus-responsive magnetic nanoparticles
　本発明は、刺激応答性磁性ナノ粒子を用いた遺伝子検査方法に関する。 The present invention relates to a genetic testing method using stimulus-responsive magnetic nanoparticles.
　核酸を含む試料から核酸を精製することによって、遺伝子の解析や同定をすることは、衛生検査、臨床検査、および研究の分野において頻繁に行われている。 Analyzing and identifying genes by purifying nucleic acids from samples containing nucleic acids is frequently performed in the fields of hygiene tests, clinical tests, and research.
　例えば、種々の動物の消化管に寄生する原虫であるクリプトスポリジウムのオーシストは強い塩素耐性を示すために、しばしば水道を介した集団下痢症を引き起こしている。このことからクリプトスポリジウムは、臨床上あるいは公衆衛生学的に現在最も注目される原虫となっている。 For example, oocysts of Cryptosporidium, a protozoan that parasitizes the digestive tract of various animals, exhibit strong chlorine tolerance, often causing mass diarrhea via water. Therefore, Cryptosporidium has become the most prominent protozoa currently in clinical or public health terms.
　クリプトスポリジウムの検出は、通常、顕微鏡下での目視により行われているが、かかる方法は、操作が煩雑で迅速性が低く、かつ検出率も低いため、検出に多大な労力を必要とする。
　従って、近年、クリプトスポリジウムの研究にも遺伝子学的手法が用いられるようになり、種の同定や型別を比較的容易に行うことができるようになってきている。 The detection of Cryptosporidium is usually performed by visual observation under a microscope. However, this method requires a lot of labor for detection because of complicated operation, low speed, and low detection rate.
Therefore, in recent years, genetic techniques have also been used for the study of Cryptosporidium, and it has become relatively easy to identify and type species.
　また、近年、遺伝子工学の発展により種々の生物の遺伝子配列が解明されつつあり、これら解明された遺伝子の機能、生物学的な役割を解明することは重要である。さらに、このような遺伝子の機能解析において、生物の各組織内でいかなる遺伝子が発現されているかを知ることは極めて重要である。例えば、こうした組織内での遺伝子発現を解析する手法の一つとして、組織からｍＲＮＡを回収し、そのｍＲＮＡを解析することが行われている。 In recent years, gene sequences of various organisms are being elucidated by the development of genetic engineering, and it is important to elucidate the functions and biological roles of these elucidated genes. Furthermore, in such functional analysis of genes, it is extremely important to know what genes are expressed in each tissue of an organism. For example, as one technique for analyzing gene expression in such a tissue, mRNA is collected from the tissue and the mRNA is analyzed.
　従来、環境・生体試料等からの核酸を分離する方法として磁気ビーズ法が用いられている（特許文献１、非特許文献１、２）。磁気ビーズ法に用いられる磁性微粒子は、磁気ビーズに磁気分離能を持たせるために、粒径をマイクロサイズに設計した磁性微粒子（例えば、ダイナビーズ（Ｄｙｎａｂｅａｄｓ）（登録商標））が用いられている。 Conventionally, the magnetic bead method has been used as a method for separating nucleic acids from the environment and biological samples (Patent Document 1, Non-Patent Documents 1 and 2). As the magnetic fine particles used in the magnetic bead method, magnetic fine particles (for example, Dynabeads (registered trademark)) whose particle size is designed to be micro size are used in order to give the magnetic beads magnetic separation ability. .
日本国特開平９－１９２９２号公報Japanese Laid-Open Patent Publication No. 9-19292
　しかしながら、特許文献１に記載の磁性微粒子を用いて磁気ビーズ法を利用して核酸（ＤＮＡ／ＲＮＡ）の回収をした場合、核酸増幅を実施する前に、核酸と磁性微粒子とを分離することにより核酸の精製を行う必要があり、作業が煩雑となっていた。 However, when nucleic acid (DNA / RNA) is recovered using the magnetic bead method using the magnetic fine particles described in Patent Document 1, the nucleic acid and magnetic fine particles are separated before performing nucleic acid amplification. It was necessary to purify the nucleic acid, and the work was complicated.
　そこで本発明は、被検試料の種別を問わず、また慎重な取り扱いを有する試薬を使わずに簡便に標的の核酸（ＤＮＡ／ＲＮＡ）の回収を行うことができ、且つ、得られた核酸を、その後の核酸分離または核酸精製等の煩雑な工程を必要とすることなく、そのまま逆転写反応及び／又は核酸増幅を行うことができる、新規な遺伝子検査方法を提供することを課題とする。 Therefore, the present invention can easily recover a target nucleic acid (DNA / RNA) regardless of the type of test sample and without using a carefully handled reagent. Therefore, an object of the present invention is to provide a novel genetic test method capable of performing reverse transcription reaction and / or nucleic acid amplification as it is without requiring complicated steps such as subsequent nucleic acid separation or nucleic acid purification.
　本発明者らは、標的の核酸に対する親和性物質が結合している刺激応答性磁性ナノ粒子を用いて核酸を回収し、非イオン性界面活性剤の存在下で核酸増幅を行うことで、刺激応答性磁性ナノ粒子を含んだ状態で逆転写反応及び／又は核酸増幅試験に課すことができることを見出し、本発明を完成するに至った。
　具体的には、本発明は以下の構成を有する。 The present inventors collect nucleic acids using stimulus-responsive magnetic nanoparticles to which an affinity substance for a target nucleic acid is bound, and perform nucleic acid amplification in the presence of a nonionic surfactant, thereby stimulating It has been found that it can be subjected to a reverse transcription reaction and / or a nucleic acid amplification test in a state in which responsive magnetic nanoparticles are contained, and the present invention has been completed.
Specifically, the present invention has the following configuration.
１．被検試料中の核酸を回収し、核酸増幅を行う遺伝子検査方法であって、以下の工程（１）～（４）を含む遺伝子検査方法。
（１）刺激応答性磁性ナノ粒子及び標的の核酸に対する親和性物質を有する吸着材と前記核酸との結合体を、被検試料を含む水溶液中で生成させる工程
（２）刺激応答性磁性ナノ粒子が凝集する条件下におき、前記結合体を凝集させる工程
（３）凝集した結合体を磁石で回収する工程
（４）回収した結合体を、非イオン性界面活性剤の存在下で核酸増幅を行う工程
２．前記非イオン性界面活性剤が、ポリオキシエチレン誘導体である前記１に記載の遺伝子検査方法。
３．前記ポリオキシエチレン誘導体が、ポリオキシエチレンソルビタンモノラウラート、ポリオキシエチレンソルビタンモノオレアート及びポリオキシエチレンオクチルフェニルエーテルからなる群より選ばれる少なくとも１である前記２に記載の遺伝子検査方法。
４．前記核酸増幅を行う工程における、前記非イオン性界面活性剤の濃度が、０．１～５質量％である、前記１～３のいずれか１に記載の遺伝子検査方法。
５．前記核酸増幅を行う工程で使用する核酸増幅酵素が、Ｔａｑポリメラーゼ、Ｂｓｔポリメラーゼ、又はＡａｃポリメラーゼのいずれかであることを特徴とする、前記１～４のいずれか１に記載の遺伝子検査方法。 1. A genetic test method for recovering nucleic acid in a test sample and performing nucleic acid amplification, comprising the following steps (1) to (4):
(1) A step of generating a conjugate of the nucleic acid and the adsorbent having an affinity substance with respect to the target nucleic acid and the nucleic acid in an aqueous solution containing the test sample (2) the stimulus responsive magnetic nanoparticle A step of aggregating the conjugate (3) a step of recovering the aggregated conjugate with a magnet (4) a nucleic acid amplification of the recovered conjugate in the presence of a nonionic surfactant Step 2 to perform 2. The genetic testing method according to 1 above, wherein the nonionic surfactant is a polyoxyethylene derivative.
3. 3. The genetic test method according to 2 above, wherein the polyoxyethylene derivative is at least 1 selected from the group consisting of polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate and polyoxyethylene octylphenyl ether.
4). 4. The genetic testing method according to any one of 1 to 3, wherein the concentration of the nonionic surfactant in the nucleic acid amplification step is 0.1 to 5% by mass.
5. 5. The gene testing method according to any one of 1 to 4, wherein the nucleic acid amplification enzyme used in the nucleic acid amplification step is Taq polymerase, Bst polymerase, or Aac polymerase.
　本発明によれば、被検試料の種別を問わず、また慎重な取り扱いを有する試薬を使わずに簡便に標的の核酸（ＤＮＡ／ＲＮＡ）の回収を行うことができる。さらに、得られた核酸を、その後の核酸分離または核酸精製等の煩雑な工程を必要とすることなく、そのまま逆転写反応及び／又は核酸増幅を行うことができる。 According to the present invention, the target nucleic acid (DNA / RNA) can be easily recovered regardless of the type of the test sample and without using a carefully handled reagent. Furthermore, the reverse transcription reaction and / or nucleic acid amplification of the obtained nucleic acid can be carried out as it is without requiring complicated steps such as subsequent nucleic acid separation or nucleic acid purification.
図１は、本発明の一実施形態に係る方法における吸着材の概略構成図である。FIG. 1 is a schematic configuration diagram of an adsorbent in a method according to an embodiment of the present invention. 図２は、非イオン性界面活性剤添加による、ＴＭ－ＡＣの濃度がＰＣＲ阻害軽減効果を示すグラフである。FIG. 2 is a graph showing the effect of reducing the inhibition of PCR by the concentration of TM-AC by adding a nonionic surfactant.
　以下、本発明の一実施形態について説明する。
　本発明は、被検試料中の核酸を回収し、核酸増幅を行う遺伝子検査方法であって、以下の工程（１）～（４）を含む遺伝子検査方法である。
（１）刺激応答性磁性ナノ粒子及び標的の核酸に対する親和性物質を有する吸着材と前記核酸との結合体を、被検試料を含む水溶液中で生成させる工程
（２）刺激応答性磁性ナノ粒子が凝集する条件下におき、前記結合体を凝集させる工程
（３）凝集した結合体を磁石で回収する工程
（４）回収した結合体を、非イオン性界面活性剤の存在下で核酸増幅を行う工程 Hereinafter, an embodiment of the present invention will be described.
The present invention is a genetic test method for recovering nucleic acid in a test sample and performing nucleic acid amplification, and includes the following steps (1) to (4).
(1) A step of generating a conjugate of the nucleic acid and the adsorbent having an affinity substance with respect to the target nucleic acid and the nucleic acid in an aqueous solution containing the test sample (2) the stimulus responsive magnetic nanoparticle A step of aggregating the conjugate (3) a step of recovering the aggregated conjugate with a magnet (4) a nucleic acid amplification of the recovered conjugate in the presence of a nonionic surfactant Process to perform
　以下、各工程について説明する。
　（１）刺激応答性磁性ナノ粒子及び標的の核酸に対する親和性物質を有する吸着材と前記核酸との結合体を、被検試料を含む水溶液中で生成させる工程 Hereinafter, each step will be described.
(1) A step of generating a conjugate of the adsorbent having stimuli-responsive magnetic nanoparticles and an affinity substance for the target nucleic acid and the nucleic acid in an aqueous solution containing the test sample.
　本発明の遺伝子検査方法は、被検試料から核酸を回収し、当該核酸を増幅することにより、遺伝子検査を行うものである。本発明においては、まず、被検試料から核酸を回収する工程を有する。本工程は、核酸回収の第１段階として、吸着材と核酸の結合体を生成させる工程である。 The genetic testing method of the present invention is to perform genetic testing by collecting nucleic acid from a test sample and amplifying the nucleic acid. In the present invention, first, there is a step of recovering a nucleic acid from a test sample. This step is a step of generating an adsorbent / nucleic acid conjugate as the first stage of nucleic acid recovery.
　（被検試料）
　被検試料としては特に限定されず、例えば、動物（ヒトを含む）、植物、微生物（原虫、原生動物等も含む）等の生物を対象とすることが可能である。また、被検試料としては、例えば、クリプトスポリジウムのようなオーシストを形成した原虫等であっても、本発明の被検試料として適用することができる。被検試料としては他にも、ウイルス、細菌、真菌等が挙げられる。 (Test sample)
The test sample is not particularly limited, and for example, organisms such as animals (including humans), plants, microorganisms (including protozoa and protozoa) can be targeted. Moreover, as a test sample, for example, even a protozoan that forms oocysts such as Cryptosporidium can be applied as the test sample of the present invention. Other test samples include viruses, bacteria, fungi, and the like.
　（核酸）
　本発明における、「核酸」とは、ＤＮＡ、ＲＮＡ及びこれらの誘導体を含む概念として用いられているものである。
　例えば、動物由来の被検試料からの核酸としては、血清、血漿等の血液成分からの血中遊離ＤＮＡ、ウイルスＤＮＡ等のＤＮＡ、血中遊離メッセンジャーＲＮＡ、ウイルスＲＮＡ等のＲＮＡ、原虫・細菌由来のＤＮＡ、リボソームＲＮＡ、メッセンジャーＲＮＡ等が挙げられる。 (Nucleic acid)
In the present invention, “nucleic acid” is used as a concept including DNA, RNA, and derivatives thereof.
For example, nucleic acids from animal-derived test samples include blood free DNA from blood components such as serum and plasma, DNA such as viral DNA, blood free messenger RNA, RNA such as viral RNA, protozoa and bacteria-derived DNA, ribosomal RNA, messenger RNA and the like.
　（吸着材）
　本発明において吸着材は、核酸を回収するために用いることができる。吸着材は、刺激応答性磁性ナノ粒子及び核酸に対する親和性物質を有し、両者が結合してなるものである。 (Adsorbent)
In the present invention, the adsorbent can be used for recovering nucleic acids. The adsorbent has an affinity substance for stimulus-responsive magnetic nanoparticles and nucleic acids, and is formed by binding both.
　（刺激応答性磁性ナノ粒子）
　本発明においては、被検試料を含む水溶液中の核酸の回収に刺激応答性磁性ナノ粒子を使用する。刺激応答性磁性ナノ粒子を使用することによって、後述する核酸増幅の際に、核酸と刺激応答性磁性ナノ粒子とを分離することによる核酸の精製を行う必要がなく、簡易に検出対象の検出を行うことができる。 (Stimulus-responsive magnetic nanoparticles)
In the present invention, stimulus-responsive magnetic nanoparticles are used for recovery of nucleic acids in an aqueous solution containing a test sample. By using stimulation-responsive magnetic nanoparticles, it is not necessary to purify nucleic acids by separating nucleic acids from stimulation-responsive magnetic nanoparticles during nucleic acid amplification, which will be described later. It can be carried out.
　本発明で用いられる刺激応答性磁性ナノ粒子は、刺激応答性ポリマーと磁性物質（磁性微粒子）とを用いて得られる。この刺激応答性ポリマーは、外的な刺激に応答して構造変化を起こし、凝集及び分散を調整できる磁性ナノ粒子である。刺激の種類は、特に限定されないが、温度、光、酸、塩基、ｐＨ若しくは電気等の様々な物理的、または化学的信号が挙げられる。 The stimulus-responsive magnetic nanoparticles used in the present invention are obtained using a stimulus-responsive polymer and a magnetic substance (magnetic fine particles). The stimulus-responsive polymer is a magnetic nanoparticle that undergoes a structural change in response to an external stimulus and can adjust aggregation and dispersion. The type of stimulation is not particularly limited, and examples thereof include various physical or chemical signals such as temperature, light, acid, base, pH, or electricity.
　特に、刺激応答性ポリマーとしては、温度変化によって凝集及び分散可能な温度応答性ポリマーが使用できる。なお、温度応答性ポリマーとしては、下限臨界溶液温度（以下、ＬＣＳＴとも称する）を有するポリマーや、上限臨界溶液温度（以下「ＵＣＳＴ」とも称する）を有するポリマーが挙げられる。 In particular, as the stimulus-responsive polymer, a temperature-responsive polymer that can be aggregated and dispersed by temperature change can be used. Examples of the temperature responsive polymer include a polymer having a lower critical solution temperature (hereinafter also referred to as LCST) and a polymer having an upper critical solution temperature (hereinafter also referred to as “UCST”).
　本発明で用いることのできる下限臨界溶液温度を有するポリマーとしては、例えば、Ｎ－ｎ－プロピルアクリルアミド、Ｎ－イソプロピルアクリルアミド、Ｎ－エチルアクリルアミド、Ｎ、Ｎ－ジメチルアクリルアミド、Ｎ－アクリロイルピロリジン、Ｎ－アクリロイルピペリジン、Ｎ－アクリロイルモルホリン、Ｎ－ｎ－プロピルメタクリルアミド、Ｎ－イソプロピルメタクリルアミド、Ｎ－エチルメタクリルアミド、Ｎ、Ｎ－ジメチルメタクリルアミド、Ｎ－メタクリロイルピロリジン、Ｎ－メタクリロイルピペリジン、Ｎ－メタクリロイルモルホリン等のＮ置換（メタ）アクリルアミド誘導体からなるポリマー；ヒドロキシプロピルセルロース、ポリビニルアルコール部分酢化物、ポリビニルメチルエーテル、（ポリオキシエチレン－ポリオキシプロピレン）ブロックコポリマー、ポリオキシエチレンラウリルアミン等のポリオキシエチレンアルキルアミン誘導体；ポリオキシエチレンソルビタンラウレート等のポリオキシエチレンソルビタンエステル誘導体；（ポリオキシエチレンノニルフェニルエーテル）アクリレート、（ポリオキシエチレンオクチルフェニルエーテル）メタクリレート等の（ポリオキシエチレンアルキルフェニルエーテル）（メタ）アクリレート類；及び（ポリオキシエチレンラウリルエーテル）アクリレート、（ポリオキシエチレンオレイルエーテル）メタクリレート等の（ポリオキシエチレンアルキルエーテル）（メタ）アクリレート類等のポリオキシエチレン（メタ）アクリル酸エステル誘導体等が挙げられる。更に、例えば、これらのポリマー及びこれらの少なくとも２種のモノマーからなるコポリマーが挙げられる。また、例えば、Ｎ－イソプロピルアクリルアミドとＮ－ｔ－ブチルアクリルアミドのコポリマーが挙げられる。 Examples of the polymer having a lower critical solution temperature that can be used in the present invention include Nn-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, N- Acryloylpiperidine, N-acryloylmorpholine, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine, N-methacryloylmorpholine Polymers composed of N-substituted (meth) acrylamide derivatives such as: hydroxypropyl cellulose, polyvinyl alcohol partially acetylated product, polyvinyl methyl ether, (polyoxyethylene Polyoxypropylene) block copolymer, polyoxyethylene alkylamine derivatives such as polyoxyethylene laurylamine; polyoxyethylene sorbitan ester derivatives such as polyoxyethylene sorbitan laurate; (polyoxyethylene nonylphenyl ether) acrylate, (polyoxyethylene) (Polyoxyethylene alkyl phenyl ether) (meth) acrylates such as octylphenyl ether) methacrylate; and (Polyoxyethylene alkyl ether) (meta) such as (polyoxyethylene lauryl ether) acrylate and (polyoxyethylene oleyl ether) methacrylate ) Polyoxyethylene (meth) acrylic acid ester derivatives such as acrylates. Furthermore, for example, a copolymer composed of these polymers and at least two kinds of these monomers can be mentioned. Further, for example, a copolymer of N-isopropylacrylamide and Nt-butylacrylamide can be mentioned.
　（メタ）アクリルアミド誘導体を含むポリマーを使用する場合、このポリマーにその他の共重合可能なモノマーを、下限臨界溶液温度を有する範囲で共重合してもよい。本発明では、なかでも、Ｎ－ｎ－プロピルアクリルアミド、Ｎ－イソプロピルアクリルアミド、Ｎ－エチルアクリルアミド、Ｎ、Ｎ－ジメチルアクリルアミド、Ｎ－アクリロイルピロリジン、Ｎ－アクリロイルピペリジン、Ｎ－アクリロイルモルホリン、Ｎ－ｎ－プロピルメタクリルアミド、Ｎ－イソプロピルメタクリルアミド、Ｎ－エチルメタクリルアミド、Ｎ、Ｎ－ジメチルメタクリルアミド、Ｎ－メタクリロイルピロリジン、Ｎ－メタクリロイルピペリジン、Ｎ－メタクリロイルモルホリンからなる群から選ばれる少なくとも１種のモノマーからなるポリマー又はＮ－イソプロピルアクリルアミドとＮ－ｔ－ブチルアクリルアミドのコポリマーが好ましく利用できる。 When a polymer containing a (meth) acrylamide derivative is used, another copolymerizable monomer may be copolymerized with this polymer within a range having a lower critical solution temperature. In the present invention, among others, Nn-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, N-acryloylpiperidine, N-acryloylmorpholine, Nn- From at least one monomer selected from the group consisting of propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine, N-methacryloylmorpholine Or a copolymer of N-isopropylacrylamide and Nt-butylacrylamide can be preferably used.
　本発明で用いることのできる上限臨界溶液温度を有するポリマーとしては、例えば、アクロイルグリシンアミド、アクロイルニペコタミド、アクリロイルアスパラギンアミド及びアクリロイルグルタミンアミド等からなる群から選ばれる少なくとも１種のモノマーからなるポリマーが挙げられる。また、これらの少なくとも２種のモノマーからなるコポリマーであってもよい。これらのポリマーには、アクリルアミド、アセチルアクリルアミド、ビオチノールアクリレート、Ｎ－ビオチニル－Ｎ’－メタクロイルトリメチレンアミド、アクロイルザルコシンアミド、メタクリルザルコシンアミド、アクロイルメチルウラシル等、その他の共重合可能なモノマーを、上限臨界溶液温度を有する範囲で共重合してもよい。 Examples of the polymer having an upper critical solution temperature that can be used in the present invention include, for example, at least one monomer selected from the group consisting of acryloylglycinamide, acryloylnipecotamide, acryloylasparaginamide, acryloylglutamineamide, and the like. The polymer which becomes is mentioned. Moreover, the copolymer which consists of these at least 2 types of monomers may be sufficient. These polymers include other copolymers such as acrylamide, acetylacrylamide, biotinol acrylate, N-biotinyl-N'-methacryloyl trimethylene amide, acroyl sarcosine amide, methacryl sarcosine amide, acroyl methyl uracil, etc. Possible monomers may be copolymerized in a range having an upper critical solution temperature.
　また、本発明では刺激応答性ポリマーとして、ｐＨ変化によって凝集及び分散可能なｐＨ応答性ポリマーが利用できる。
　このようなｐＨ応答性ポリマーとしては、例えば、カルボキシル、リン酸、スルホニル、アミノ等の基を官能基として含有するポリマーが挙げられる。より具体的には、例えば、アクリル酸、メタクリル酸、マレイン酸、ビニルスルホン酸、ビニルベンゼンスルホン酸、ホスホリルエチル（メタ）アクリレート、アミノエチルメタクリレート、アミノプロピル（メタ）アクリルアミド、ジメチルアミノプロピル（メタ）アクリルアミドまたはこれらの塩を共重合成分として含むポリマーが挙げられる。 In the present invention, a pH-responsive polymer that can be aggregated and dispersed by pH change can be used as the stimulus-responsive polymer.
Examples of such a pH-responsive polymer include a polymer containing a group such as carboxyl, phosphoric acid, sulfonyl, and amino as a functional group. More specifically, for example, acrylic acid, methacrylic acid, maleic acid, vinyl sulfonic acid, vinyl benzene sulfonic acid, phosphoryl ethyl (meth) acrylate, aminoethyl methacrylate, aminopropyl (meth) acrylamide, dimethylaminopropyl (meth) Examples include polymers containing acrylamide or a salt thereof as a copolymerization component.
　磁性物質である磁性微粒子は、例えば、多価アルコールとマグネタイトとで構成される。 Magnetic fine particles, which are magnetic substances, are composed of, for example, polyhydric alcohol and magnetite.
　多価アルコールは、構成単位に水酸基を少なくとも２個有し、且つ鉄イオンと結合可能なアルコール構造体であれば特に限定されない。例えば、デキストラン、ポリビニルアルコール、マンニトール、ソルビトール及びシクロデキストリンが挙げられる。例えば日本国特開２００５－８２５３８号公報には、デキストランを用いた微粒子状の磁性物質の製造方法が開示されている。また、グリシジルメタクリレート重合体のようにエポキシを有し、開環後多価アルコール構造体を形成する化合物も使用できる。 The polyhydric alcohol is not particularly limited as long as it is an alcohol structure having at least two hydroxyl groups in a structural unit and capable of binding to iron ions. Examples include dextran, polyvinyl alcohol, mannitol, sorbitol and cyclodextrin. For example, Japanese Patent Application Laid-Open No. 2005-82538 discloses a method for producing a particulate magnetic material using dextran. Moreover, the compound which has an epoxy like a glycidyl methacrylate polymer and forms a polyhydric alcohol structure after ring-opening can also be used.
　このような多価アルコールを用いて調製された微粒子状の磁性物質（磁性微粒子）は、良好な分散性を有するように、その平均粒径が０．９ｎｍ以上１０００ｎｍ未満であることが好ましい。平均粒径は、特に目的とする検出対象の検出感度を高めるためには、２．９ｎｍ以上２００ｎｍ未満であることが好ましい。また、磁性微粒子の平均粒径が前記範囲であることによって、液体中での分散性が良好となる。その結果、核酸増幅試験の際に反応液中で磁性微粒子が沈降せず適度に分散し、効率よく逆転写反応や核酸増幅試験を行うことができる。 The fine particle magnetic material (magnetic fine particles) prepared using such a polyhydric alcohol preferably has an average particle size of 0.9 nm or more and less than 1000 nm so as to have good dispersibility. The average particle size is preferably 2.9 nm or more and less than 200 nm, in particular, in order to increase the detection sensitivity of the target detection target. Moreover, the dispersibility in a liquid becomes favorable because the average particle diameter of a magnetic fine particle is the said range. As a result, during the nucleic acid amplification test, the magnetic fine particles do not settle in the reaction solution and are appropriately dispersed, and the reverse transcription reaction and the nucleic acid amplification test can be performed efficiently.
　（親和性物質）
　本発明で用いられる親和性物質としては、核酸に対して結合するものであれば特に限定されるものではない。例えば、核酸、アプタマー、カチオン性官能基を有する物質（例えば、１級アミノ基、２級アミノ基、３級アミノ基、４級アンモニウム基またはグアニジノ基を有する物質）、アニオン性官能基を有する物質、標的物質と相互作用する蛋白質（例えば、酵素、レセプター）、キレート剤などが挙げられる。 (Affinity substance)
The affinity substance used in the present invention is not particularly limited as long as it binds to a nucleic acid. For example, nucleic acids, aptamers, substances having a cationic functional group (for example, substances having a primary amino group, secondary amino group, tertiary amino group, quaternary ammonium group or guanidino group), substances having an anionic functional group And proteins that interact with the target substance (eg, enzymes, receptors), chelating agents, and the like.
　（刺激応答性磁性ナノ粒子と親和性物質との結合）
　刺激応答性磁性ナノ粒子と親和性物質との結合方法は、特に限定されない。例えば、刺激応答性磁性ナノ粒子側（例えば刺激応答性ポリマー部分）及び親和性物質側の双方に、互いに親和性の物質（例えば、ビオチン及びストレプトアビジンまたはアビジン）を結合させ、これら物質を介して刺激応答性磁性ナノ粒子と親和性物質を結合させる。 (Binding of stimuli-responsive magnetic nanoparticles and affinity substances)
The binding method between the stimulus-responsive magnetic nanoparticles and the affinity substance is not particularly limited. For example, substances having affinity (for example, biotin and streptavidin or avidin) are bound to both the stimulus-responsive magnetic nanoparticle side (for example, the stimulus-responsive polymer portion) and the affinity substance side, and through these substances A stimulus-responsive magnetic nanoparticle is bound to an affinity substance.
　具体的には、刺激応答性ポリマーへのビオチンの結合は、国際公開第２００１／００９１４１号に記載されているように、ビオチン等をメタクリルやアクリル等の重合性官能基と結合させて付加重合性モノマーとし、他のモノマーと共重合することにより行い、そこにストレプトアビジンを固定化する。また、親和性物質へのビオチン等の標識は常法に従って行う。ビオチンで標識された親和性物質とストレプトアビジン固定化刺激応答性ポリマーとを混合すると、ビオチンとストレプトアビジンとの結合を介して、刺激応答性ポリマー及び親和性物質が結合する。 Specifically, biotin is bonded to the stimulus-responsive polymer by adding biotin or the like to a polymerizable functional group such as methacryl or acryl as described in International Publication No. 2001/009141. A monomer is used and copolymerized with other monomers to immobilize streptavidin. In addition, labeling of the affinity substance with biotin or the like is performed according to a conventional method. When the affinity substance labeled with biotin and the streptavidin-immobilized stimulus-responsive polymer are mixed, the stimulus-responsive polymer and the affinity substance are bound via the binding between biotin and streptavidin.
　具体的には、図１に示されるように、刺激応答性磁性ナノ粒子１は微粒子状の磁性物質２を含み、この磁性物質２の表面に刺激応答性ポリマー３が結合されている。刺激応答性ポリマー３にはストレプトアビジン４が結合しており、ビオチン５を介して検出対象７に対する親和性物質６が結合する。 Specifically, as shown in FIG. 1, the stimulus-responsive magnetic nanoparticle 1 includes a particulate magnetic substance 2, and a stimulus-responsive polymer 3 is bonded to the surface of the magnetic substance 2. Streptavidin 4 is bound to the stimulus-responsive polymer 3, and an affinity substance 6 for the detection target 7 is bound via biotin 5.
　なお、本明細書では、刺激応答性ポリマーと微粒子状の磁性物質とが結合した材料を刺激応答性磁性ナノ粒子と記し、その刺激が熱の場合には、熱応答性磁性ナノ粒子と記している。 In this specification, a material in which a stimulus-responsive polymer and a particulate magnetic substance are combined is referred to as a stimulus-responsive magnetic nanoparticle, and when the stimulus is heat, it is referred to as a thermoresponsive magnetic nanoparticle. Yes.
　微粒子状の磁性物質と刺激応答性ポリマーとの結合は、反応性官能基を介して結合する方法や、磁性物質中の多価アルコール上の活性水素又は多価アルコールに重合性不飽和結合を導入してグラフト重合する方法等の当技術分野で周知の方法で行ってよい（例えば、ＡＤＶ．Ｐｏｌｙｍ．Ｓｃｉ．，Ｖｏｌ．４，ｐ１１１，１９６５や、Ｊ．Ｐｏｌｙｍｅｒ　Ｓｃｉ．，Ｐａｒｔ－Ａ，３，ｐ１０３１、１９６５参照）。 The fine magnetic substance can be bonded to the stimulus-responsive polymer via a reactive functional group, or a polymerizable unsaturated bond is introduced into the active hydrogen or polyhydric alcohol on the polyhydric alcohol in the magnetic substance. The graft polymerization may be performed by a method known in the art such as graft polymerization (for example, ADV.Polym.Sci., Vol.4, p111, 1965, J. Polymer Sci., Part-A, 3, p1031, 1965).
　（水溶液中における吸着材と前記検出対象の結合体の生成）
　上述した吸着材と核酸との結合体は、水溶液中で吸着材と核酸を任意の方法で混合させることによって、吸着材中の親和性物質と核酸が結合し、吸着材と核酸の結合体が生成する。親和性物質と核酸との結合は、特異的な結合ないし吸着を意味し、必ずしも共有結合である必要はなく、イオン的、生化学的親和性（アフィニティー）を利用した結合であってもよい。 (Generation of the adsorbent and the conjugate to be detected in an aqueous solution)
The adsorbent / nucleic acid conjugate described above is obtained by mixing the adsorbent and nucleic acid in an aqueous solution by any method, whereby the affinity substance in the adsorbent and the nucleic acid are combined, and the adsorbent / nucleic acid conjugate is formed. Generate. The binding between the affinity substance and the nucleic acid means specific binding or adsorption, and is not necessarily a covalent binding, and may be binding utilizing ionic or biochemical affinity (affinity).
　混合操作は、適当なバッファー中で、吸着材と核酸が接触し得るならば特に制限はない。例えば、核酸を含む被検試料及び吸着材が供されたチューブを軽く転倒撹拌または振とうさせる程度で十分であり、例えば市販のボルテックスミキサー等を用いて混合する操作等が挙げられる。 The mixing operation is not particularly limited as long as the adsorbent and the nucleic acid can come into contact with each other in an appropriate buffer. For example, it is sufficient to lightly invert or shake the test sample containing nucleic acid and the tube provided with the adsorbent, and examples thereof include an operation of mixing using a commercially available vortex mixer or the like.
　（２）刺激応答性磁性ナノ粒子が凝集する条件下におき、結合体を凝集させる工程
　本工程は核酸回収の第２段階として、工程（１）において核酸と吸着材の結合体が得られた後、刺激応答性ポリマーが凝集する条件下におき、結合体を凝集させる工程である。 (2) A step of aggregating the conjugate under conditions where the stimuli-responsive magnetic nanoparticles are agglomerated This step is a second step of nucleic acid recovery, and a conjugate of nucleic acid and adsorbent was obtained in step (1). Then, it is the process which puts on the conditions which a stimulus responsive polymer aggregates, and aggregates a conjugate | bonded_body.
　刺激応答性ポリマーが凝集する条件とするには、例えば熱応答性ポリマーを用いた場合、混合物の入った容器を熱応答性ポリマーの凝集する温度の恒温槽に移せばよい。熱応答性ポリマーには、上限臨界溶液温度を有するポリマーと、下限臨界溶液温度を有するポリマーの２種類がある。 For the conditions under which the stimulus-responsive polymer aggregates, for example, when a thermoresponsive polymer is used, the container containing the mixture may be transferred to a thermostatic bath at a temperature at which the thermoresponsive polymer aggregates. There are two types of thermoresponsive polymers: polymers having an upper critical solution temperature and polymers having a lower critical solution temperature.
　例えば、ＬＣＳＴが３７℃である下限臨界溶液温度を有するポリマーを用いた場合には、混合物の入った容器を３７℃以上の恒温槽に移すことで、熱応答性ポリマーを凝集させることができる。また、ＵＣＳＴが５℃である上限臨界溶液温度を有するポリマーを用いた場合には、混合物の入った容器を５℃未満の恒温槽に移すことで、熱応答性ポリマーを凝集させることができる。 For example, when a polymer having a lower critical solution temperature with an LCST of 37 ° C. is used, the thermoresponsive polymer can be agglomerated by moving the container containing the mixture to a constant temperature bath at 37 ° C. or higher. When a polymer having an upper critical solution temperature with a UCST of 5 ° C. is used, the thermoresponsive polymer can be agglomerated by transferring the container containing the mixture to a thermostatic bath of less than 5 ° C.
　ここで、下限臨界溶液温度及び上限臨界溶液温度は例えば以下のように決定する。まず、試料を吸光光度計のセルに入れ、１℃／分の速度で試料を昇温する。この間、５５０ｎｍにおける透過率変化を記録する。ここで、ポリマーが透明に溶解しているときの透過率を１００％、完全に凝集したときの透過率を０％としたとき、透過率が５０％になるときの温度をＬＣＳＴとして求める。ＵＣＳＴの場合は、１℃／分の速度で試料を冷却し、以下、ＬＣＳＴ同様の方法で求める。 Here, the lower critical solution temperature and the upper critical solution temperature are determined as follows, for example. First, a sample is put into a cell of an absorptiometer and the sample is heated at a rate of 1 ° C./min. During this time, the change in transmittance at 550 nm is recorded. Here, when the transmittance when the polymer is transparently dissolved is 100% and when the transmittance when the polymer is completely aggregated is 0%, the temperature at which the transmittance is 50% is obtained as LCST. In the case of UCST, the sample is cooled at a rate of 1 ° C./min, and thereafter obtained by the same method as LCST.
　また、ｐＨ応答性ポリマーを用いた場合、混合物の入った容器に酸溶液又はアルカリ溶液を加えればよい。具体的には、ｐＨ応答性ポリマーが構造変化を起こすｐＨ範囲の外にある分散している混合物の入った容器に、酸溶液又はアルカリ溶液を加え、容器内をｐＨ応答性ポリマーが構造変化を起こすｐＨ範囲に変更すればよい。例えば、ｐＨ５以下で凝集、ｐＨ５超で分散するｐＨ応答性ポリマーを用いた場合、ｐＨ５超で分散している混合物の入った容器に、ｐＨが５以下になるように酸溶液を加えればよい。また、ｐＨ１０以上で凝集、ｐＨ１０未満で分散するｐＨ応答性ポリマーを用いた場合、ｐＨ１０未満で分散している混合物の入った容器に、ｐＨが１０以上になるようにアルカリ溶液を加えればよい。ｐＨ応答性ポリマーが構造変化を起こすｐＨは、特に限定されないが、ｐＨ４～１０が好ましく、ｐＨ５～９であることがさらに好ましい。 Further, when a pH responsive polymer is used, an acid solution or an alkali solution may be added to a container containing the mixture. Specifically, an acid solution or an alkaline solution is added to a container containing a dispersed mixture outside the pH range where the pH-responsive polymer undergoes a structural change, and the pH-responsive polymer undergoes a structural change in the container. What is necessary is just to change to the pH range to raise. For example, when a pH-responsive polymer that aggregates at a pH of 5 or less and disperses at a pH of more than 5 is used, an acid solution may be added to a container containing a mixture dispersed at a pH of more than 5 so that the pH is 5 or less. When a pH-responsive polymer that aggregates at pH 10 or higher and disperses at a pH lower than 10 is used, an alkaline solution may be added to a container containing a mixture dispersed at a pH lower than 10 so that the pH becomes 10 or higher. The pH at which the pH-responsive polymer undergoes a structural change is not particularly limited, but is preferably pH 4 to 10, and more preferably pH 5 to 9.
　また、光応答性ポリマーを用いた場合、混合物の入った容器にポリマーを凝集できる波長の光を照射すればよい。凝集させるための好ましい光は、光応答性ポリマーに含まれる光応答性官能基の種類及び構造により異なるが、一般に波長１９０～８００ｎｍの紫外光又は可視光が好適に使用できる。このとき、強度は０．１～１０００ｍＷ／ｃｍ２が好ましい。 Further, when a photoresponsive polymer is used, light having a wavelength capable of aggregating the polymer may be irradiated to a container containing the mixture. The preferred light for aggregation varies depending on the type and structure of the photoresponsive functional group contained in the photoresponsive polymer, but in general, ultraviolet light or visible light having a wavelength of 190 to 800 nm can be suitably used. At this time, the strength is preferably 0.1 to 1000 mW / cm 2 .
　なお、光応答性ポリマーは、測定精度を向上できる点で、濁度の測定に用いられる光が照射された際に分散を生じにくいもの、換言すれば凝集するものであることが好ましい。光応答性ポリマーとして、濁度の測定に用いられる光が照射された際に分散を生じるものを用いる場合、照射時間を短縮することで測定精度を向上できる。 It should be noted that the photoresponsive polymer is preferably one that is less likely to cause dispersion when irradiated with light used for turbidity measurement, in other words, that it can aggregate in that it can improve measurement accuracy. When using a photoresponsive polymer that generates dispersion when irradiated with light used for turbidity measurement, the measurement accuracy can be improved by shortening the irradiation time.
　（３）凝集した結合体を磁石で回収する工程
　本工程は検出対象回収の最終段階として、工程（２）で凝集した結合体を磁石で分離し、検出対象を回収する工程である。磁性粒子を含有する結合体に磁力を付加することで、凝集した結合体を混合液中から分離し、回収する。これにより、結合体が非凝集状態の磁性物質を含む夾雑物から分離され、検出対象の回収が可能となる。 (3) Step of recovering aggregated conjugate with magnet This step is a step of recovering the detection target by separating the aggregate aggregated in step (2) with a magnet as the final stage of detection target recovery. By applying a magnetic force to the conjugate containing magnetic particles, the aggregated conjugate is separated from the mixed solution and recovered. As a result, the conjugate is separated from the contaminants containing the non-aggregated magnetic substance, and the detection target can be recovered.
　例えば、核酸と吸着材との結合を適当なチューブ内で行った場合、チューブの側壁に磁石を外側から近づけることによって吸着材をチューブの側壁近傍に保持しつつ、チューブ内から上澄み部分となる液体を排出することによって、核酸が結合した吸着材を分離することができる。 For example, when binding of nucleic acid and adsorbent is performed in a suitable tube, the adsorbent is held near the side wall of the tube by bringing the magnet close to the side wall of the tube from the outside, and the liquid that becomes the supernatant part from the inside of the tube By discharging the adsorbent, the adsorbent to which the nucleic acid is bound can be separated.
　核酸が結合した吸着材の分離に用いる磁石等の磁力は、用いる磁性物質の有する磁力の大きさによって異なる。磁力は、目的の磁性物質を磁集可能な程度の磁力を適宜使用できる。磁石の素材としては、例えば、上述した磁性物質の素材で構成されたものを使用することができる。例えば、ネオジム磁石（株式会社二六製作所製）等が利用できる。ネオジム磁石の磁力は、３８００ガウス以上が好ましい。 The magnetic force of a magnet or the like used for separating the adsorbent to which nucleic acid is bound varies depending on the magnitude of the magnetic force of the magnetic substance used. As the magnetic force, a magnetic force capable of collecting the target magnetic substance can be appropriately used. As the material of the magnet, for example, a material made of the above-described magnetic material can be used. For example, a neodymium magnet (manufactured by 26 Co., Ltd.) can be used. The magnetic force of the neodymium magnet is preferably 3800 gauss or more.
　（４）回収した結合体を、非イオン性界面活性剤の存在下で核酸増幅を行う工程
　本工程は、上記工程で回収された結合体中の核酸を試料として核酸増幅を行う工程である。上述したように、吸着材中の刺激応答性磁性ナノ粒子は、反応液中の刺激応答性磁性ナノ粒子の濃度が高いと、逆転写反応や核酸増幅を阻害しうる。そのためこれまでは逆転写反応や核酸増幅試験の前に核酸と磁性微粒子とを分離することにより核酸の精製を行う必要があった。しかしながら本発明では、非イオン性界面活性剤の存在下で核酸増幅を行うことによって、刺激応答性磁性ナノ粒子による逆転写反応及び核酸増幅の阻害を軽減することができる。 (4) Step of performing nucleic acid amplification on the recovered conjugate in the presence of a nonionic surfactant This step is a step of performing nucleic acid amplification using the nucleic acid in the conjugate recovered in the above step as a sample. As described above, the stimulus-responsive magnetic nanoparticles in the adsorbent can inhibit reverse transcription reaction and nucleic acid amplification when the concentration of the stimulus-responsive magnetic nanoparticles in the reaction solution is high. For this reason, it has been necessary to purify the nucleic acid by separating the nucleic acid and the magnetic fine particles before the reverse transcription reaction or the nucleic acid amplification test. However, in the present invention, by performing nucleic acid amplification in the presence of a nonionic surfactant, it is possible to reduce inhibition of reverse transcription reaction and nucleic acid amplification by stimulus-responsive magnetic nanoparticles.
　上述したように、本工程では、検出対象の検出感度の向上のため、上記工程で回収された核酸をそのまま試料として核酸増幅を行うことが可能である。核酸がＲＮＡである場合には、逆転写反応を行いＤＮＡとしてから核酸増幅試験を行う。逆転写反応は、上記工程（１）～（３）で得られたＲＮＡを鋳型として、従来の公知の任意の方法を採用することができる。 As described above, in this step, in order to improve the detection sensitivity of the detection target, it is possible to perform nucleic acid amplification using the nucleic acid collected in the above step as it is as a sample. When the nucleic acid is RNA, a reverse transcription reaction is performed to obtain DNA, and then a nucleic acid amplification test is performed. For the reverse transcription reaction, any conventionally known method can be employed using the RNA obtained in the above steps (1) to (3) as a template.
　また、核酸増幅の方法としては、任意の方法を使用することができ、例えば、ポリメラーゼ連鎖反応（ＰＣＲ法）、ＬＡＭＰ法、鎖置換増幅、逆転写酵素鎖置換増幅、逆転写酵素ポリメラーゼ連鎖反応、逆転写ＬＡＭＰ法、核酸配列に基づく増幅、転写媒介性増幅およびローリングサークル型増幅法、ＳｍａｒｔＡｍｐ、法逆転写ＳｍａｒｔＡｍｐ法等が挙げられる。 Moreover, any method can be used as a method for nucleic acid amplification, such as polymerase chain reaction (PCR method), LAMP method, strand displacement amplification, reverse transcriptase strand displacement amplification, reverse transcriptase polymerase chain reaction, Examples include reverse transcription LAMP method, nucleic acid sequence-based amplification, transcription-mediated amplification and rolling circle amplification method, SmartAmp, and method reverse transcription SmartAmp method.
　通常、ＤＮＡの増幅は、ＰＣＲ法により行うことが一般的である。ＰＣＲの方法としては、上記工程で得られたＤＮＡ、またはＲＮＡの逆転写反応により得られるＤＮＡを鋳型として、従来の公知の任意のＰＣＲ方法を採用することができる。本発明においては、ＰＣＲ増幅時の核酸増幅酵素が、Ｔａｑポリメラーゼ、Ｂｓｔポリメラーゼ、又はＡａｃポリメラーゼのいずれかである場合には、特に、好適に利用可能である。 Usually, DNA amplification is generally performed by PCR. As the PCR method, any conventionally known PCR method can be employed using the DNA obtained in the above step or the DNA obtained by reverse transcription reaction of RNA as a template. In the present invention, when the nucleic acid amplification enzyme at the time of PCR amplification is any of Taq polymerase, Bst polymerase, or Aac polymerase, it can be suitably used.
　（非イオン性界面活性剤）
　本工程では、非イオン性界面活性剤の存在下で核酸増幅を行うことによって、刺激応答性磁性ナノ粒子による逆転写反応や核酸増幅の阻害を軽減することができる。 (Nonionic surfactant)
In this step, by performing nucleic acid amplification in the presence of a nonionic surfactant, it is possible to reduce inhibition of reverse transcription reaction and nucleic acid amplification by stimuli-responsive magnetic nanoparticles.
　非イオン性界面活性剤の存在下で核酸増幅を行うことによって、刺激応答性磁性ナノ粒子による逆転写反応や核酸増幅の阻害を軽減することができる詳細は定かではないが、刺激応答性磁性ナノ粒子表面のポリマーに非イオン性界面活性剤が作用して、ミセルを形成することによるものと推測される。 Although it is not clear that the nucleic acid amplification in the presence of a nonionic surfactant can reduce the inhibition of reverse transcription reaction and nucleic acid amplification by stimuli-responsive magnetic nanoparticles, the stimulus-responsive magnetic nanoparticle is not clear. It is presumed that the nonionic surfactant acts on the polymer on the particle surface to form micelles.
　本発明で使用できる非イオン性界面活性剤としては、例えば、ポリオキシエチレン誘導体が挙げられる。ポリオキシエチレン誘導体としては、例えば、ポリオキシエチレンソルビタンモノラウラート（Ｔｗｅｅｎ２０）、ポリオキシエチレンソルビタンモノオレアート（Ｔｗｅｅｎ８０）、ポリオキシエチレン（１０）オクチルフェニルエーテル（ＴｒｉｔｏｎＸ－１００）等が挙げられる。中でも、粘性が低くて扱いやすいという観点から、ポリオキシエチレンソルビタンモノラウラート、ポリオキシエチレン（１０）オクチルフェニルエーテル（ＴｒｉｔｏｎＸ－１００）が好ましい。 Examples of the nonionic surfactant that can be used in the present invention include polyoxyethylene derivatives. Examples of the polyoxyethylene derivative include polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene (10) octylphenyl ether (Triton X-100), and the like. Among them, polyoxyethylene sorbitan monolaurate and polyoxyethylene (10) octylphenyl ether (Triton X-100) are preferable from the viewpoint of low viscosity and easy handling.
　また、逆転写反応や核酸増幅を行う際には、非イオン性界面活性剤の濃度を、好ましくは０．１～５質量％、より好ましくは０．５～５質量％とするように希釈する。上記範囲であることによって、刺激応答性磁性ナノ粒子による核酸増幅阻害を軽減することができる。 Further, when performing reverse transcription reaction or nucleic acid amplification, the concentration of the nonionic surfactant is preferably diluted to 0.1 to 5% by mass, more preferably 0.5 to 5% by mass. . By being in the above range, inhibition of nucleic acid amplification by the stimulus-responsive magnetic nanoparticles can be reduced.
　また、逆転写反応や核酸増幅試験を好適に行うため、核酸試料中に残存する刺激応答性ナノ粒子は、好ましくは０．１～１質量％、より好ましくは０．１～０．５質量％である。上記範囲であることによって、刺激応答性磁性ナノ粒子による核酸増幅阻害を軽減することができる。 In order to perform a reverse transcription reaction and a nucleic acid amplification test suitably, the stimulus-responsive nanoparticles remaining in the nucleic acid sample are preferably 0.1 to 1% by mass, more preferably 0.1 to 0.5% by mass It is. By being in the above range, inhibition of nucleic acid amplification by the stimulus-responsive magnetic nanoparticles can be reduced.
　本発明を以下の実施例等により説明するが、これらによって制限されるものではない。
　但し、「％」は「質量％」を示す。 The present invention is illustrated by the following examples, but is not limited thereto.
However, “%” indicates “mass%”.
［実施例１］
（１）オーシスト回収用磁性ナノ粒子（ＴＭ－ＡＣ）の作製
　アビジンが結合している熱応答性磁性ナノ粒子（Ｔｈｅｒｍａ－Ｍａｘ　ＬＡ　Ａｖｉｄｉｎ，４ｇ／Ｌ，ＪＮＣ株式会社）１ｍＬとＣ．ｐａｒｖｕｍオーシストに特異的なビオチン化モノクローナル抗体（Ｃｒｙｐｔ－ａ－Ｇｌｏ　ｂｉｏｔｉｎ　ｒｅａｇｅｎｔキット（２０倍濃縮），Ｗａｔｅｒｂｏｒｎｅ社）２０μＬを混合・結合させて、オーシスト回収用磁性ナノ粒子（Ｔｈｅｒｍａ－Ｍａｘ－Ａｎｔｉ－Ｃｒｙｐｔｏｓｐｏｒｉｄｉｕｍ：ＴＭ－ＡＣ）を作製した。 [Example 1]
(1) Production of magnetic nanoparticles for recovery of oocysts (TM-AC) 1 mL of thermoresponsive magnetic nanoparticles to which avidin is bonded (Therma-Max LA Avidin, 4 g / L, JNC Corporation) and C.I. 20 μL of biotinylated monoclonal antibody specific for parvum oocysts (Crypt-a-Glo biotin reagent kit (20-fold concentrated), Waterborne) was combined and bound to magnetic nanoparticles for recovery of oocysts (Therma-Max-Anti-Cryptosporidium) : TM-AC).
（２）リアルタイムＰＣＲの実施
　ＴＭ－ＡＣの終濃度を０．１％に調製し、ＤＮＡ試料とともに、リアルタイムＰＣＲを行った。
＜ＤＮＡ試料＞
　クリプトスポリジウムのＤＮＡ試料として、人工合成ＤＮＡ（Ｃ．ｐａｒｖｕｍ　１８Ｓ　ｒＤＮＡ　ｇｅｎｅ，１９２ｂｐ）（ｔｔｔａ　ｔｇｇａａｇｇｇｔｔ　ｇｔａｔｔｔａｔｔａ　ｇａｔａａａｇａａｃ　ｃａａｔａｔａａｔｔ　ｇｇｔｇａｃｔｃａｔ　ａａｔａａｃｔｔｔａ　ｃｇｇａｔｃａｃａｔ　ｔａａａｔｇｔｇａｃ　ａｔａｔｃａｔｔｃａ　ａｇｔｔｔｃｔｇａｃ　ｃｔａｔｃａｇｃｔｔ　ｔａｇａｃｇｇｔａｇ　ｇｇｔａｔｔｇｇｃｃ　ｔａｃｃｇｔｇｇｃａ　ａｔｇａｃｇｇｇｔａ　ａｃｇｇｇｇａａｔｔ　ａｇｇｇｔｔｃｇａｔ　ｔｃｃｇｇａｇａｇｇ　ｇａｇｃｃｔｇａ：配列番号１）が挿入されたＤＮＡプラスミド溶液（ユーロフィンジェノミクス社）を用いた。 (2) Implementation of real-time PCR The final concentration of TM-AC was adjusted to 0.1%, and real-time PCR was performed together with the DNA sample.
<DNA sample>
As Cryptosporidium DNA sample, artificially synthesized DNA (C.parvum 18S rDNA gene, 192bp) (ttta tggaagggtt gtatttatta gataaagaac caatataatt ggtgactcat aataacttta cggatcacat taaatgtgac atatcattca agtttctgac ctatcagctt tagacggtag ggtattggcc taccgtggca atgacgggta acggggaatt agggttcgat tccggagagg gagcctga: SEQ ID NO: 1) has been inserted A DNA plasmid solution (Eurofin Genomics) was used.
＜ＰＣＲ条件＞
　ＰＣＲはクリプトスポリジウム１８Ｓ　ｒＤＮＡ遺伝子に特異的なプライマーセット　ｆｏｒｗａｒｄ：５’－　ＧＧＡＡＧＧＧＴＴＧＴＡＴＴＴＡＴＴＡＧＡＴＡＡＡＧＡＡＣＣ－３’（配列番号２）、ｒｅｖｅｒｓｅ：５’－　ＣＴＣＣＣＴＣＴＣＣＧＧＡＡＴＣＧＡＡ－３（配列番号３）（各５μＭ）０．８μＬとＴａｑＭａｎプローブ：ＴＣＴＧＡＣＣＺＡＴＣＡＧＣＴＴ（配列番号４）（１μＭ）２．０μＬ、ＰＣＲ試薬Ｐｒｅｍｉｘ　ＥＸ　Ｔａｑ（タカラバイオ株式会社）１０μＬ、Ｔｗｅｅｎ２０（Ｔｗｅｅｎ２０（登録商標）、Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ　Ｇｒａｄｅ、Ｐｒｏｍｅｇａ製）、リアルタイムＰＣＲ（ＬｉｇｈｔＣｙｃｌｅｒ　Ｎａｎｏ，ロシュ・ダイアグノスティックス株式会社）を用いて行った。Ｔｗｅｅｎ２０は終濃度が５％となるよう調整した。陽生対照（５×１０６ｃｏｐｉｅｓ／ｍＬ）の添加量は２μＬ、ＰＣＲの反応液量は２０μＬで行った。
　ＰＣＲ条件は「Ｈｏｌｄ；１ｃｙｃｌｅ（９５℃、３０ｓ），２Ｓｔｅｐ　ＰＣＲ；４５ｃｙｃｌｅｓ（６０℃、３０ｓ，９５℃、１０ｓ）」で行った。Ｃｔ値（Ｔｈｒｅｓｈｏｌｄ　Ｃｙｃｌｅ）はＬｉｇｈｔＣｙｃｌｅｒ　Ｎａｎｏ　ソフトウェアで解析した。 <PCR conditions>
PCR is a primer set specific for Cryptosporidium 18S rDNA gene forward: 5′-GGAAGGGTTGTATTATTATAGATAAAGAGAACC-3 ′ (SEQ ID NO: 2), reverse: 5′-CTCCCTCTCCGGGAATCGAA-3 (SEQ ID NO: 3) (each 5 μM) 0.8 μL and TaqMan Probe: TCTGACCZATCAGCTT (SEQ ID NO: 4) (1 μM) 2.0 μL, PCR reagent Premix EX Taq (Takara Bio Inc.) 10 μL, Tween 20 (Tween 20 (registered trademark), Molecular Biology Grade, manufactured by Promega), real-time PCR (LightCycC) Roche Diagnostics Inc.). Tween 20 was adjusted to a final concentration of 5%. The added amount of the positive control (5 × 10 6 copies / mL) was 2 μL, and the PCR reaction solution volume was 20 μL.
PCR conditions were “Hold; 1 cycle (95 ° C., 30 s), 2 Step PCR; 45 cycles (60 ° C., 30 s, 95 ° C., 10 s)”. The Ct value (Threshold Cycle) was analyzed with LightCycler Nano software.
［比較例１］
　比較例１は、ＰＣＲ時にＴｗｅｅｎ２０を添加しなかったこと以外は、実施例１と同様に試験を行った。
［実施例２、比較例２］
　実施例２は、ＴＭ－ＡＣの終濃度を０．５％に調製したこと以外は、実施例１と同様に試験を行った。比較例２は、ＴＭ－ＡＣの終濃度を０．５％に調製し、ＰＣＲ時にＴｗｅｅｎ２０を添加しなかったこと以外は、実施例１と同様に試験を行った。
［対照例１、対照例２］
　対照例１は、ＴＭ－ＡＣの終濃度を０％としたこと以外は、実施例１と同様に試験を行った。対照例２は、ＴＭ－ＡＣの終濃度を０％とし、ＰＣＲ時にＴｗｅｅｎ２０を添加しなかったこと以外は、実施例１と同様に試験を行った。 [Comparative Example 1]
Comparative Example 1 was tested in the same manner as in Example 1 except that Tween 20 was not added during PCR.
[Example 2, Comparative Example 2]
Example 2 was tested in the same manner as Example 1 except that the final concentration of TM-AC was adjusted to 0.5%. Comparative Example 2 was tested in the same manner as in Example 1 except that the final TM-AC concentration was adjusted to 0.5% and Tween 20 was not added during PCR.
[Control Example 1, Control Example 2]
Control 1 was tested in the same manner as Example 1 except that the final TM-AC concentration was 0%. Control Example 2 was tested in the same manner as in Example 1 except that the final concentration of TM-AC was 0% and Tween 20 was not added during PCR.
　ＴＭ－ＡＣによるＰＣＲ阻害を評価した結果を図１（白四角：Ｔｗｅｅｎ２０添加なし）に示す。縦軸のＣｔ値はＰＣＲ増幅産物がある一定量に達したときのサイクル数を表している。ＰＣＲ増幅産物の量が多いほどＣｔ値は小さくなり、増幅されない場合は検出されない。比較例１のＴＭ－ＡＣ添加濃度０．１％のＣｔ値は対照例１（ＴＭ－ＡＣ添加０％）とほぼ同じであったことから、ＰＣＲが阻害されていないことがわかる。しかし、比較例２のＴＭ－ＡＣ添加０．５％では増幅速度が大幅に遅れＣｔ値は２５．２となった。この結果より、ＰＣＲチューブ（反応液２０μＬ）にＴＭ－ＡＣを０．１μＬ添加した場合（ＴＭ－ＡＣ終濃度０．５％）、ＰＣＲが阻害されることがわかる。 The results of evaluating PCR inhibition by TM-AC are shown in FIG. 1 (white squares: no Tween 20 added). The Ct value on the vertical axis represents the number of cycles when the PCR amplification product reaches a certain amount. The greater the amount of PCR amplification product, the smaller the Ct value, and it is not detected when it is not amplified. The Ct value of the TM-AC addition concentration of 0.1% in Comparative Example 1 was almost the same as that of Control Example 1 (TM-AC addition 0%), which indicates that PCR was not inhibited. However, with the TM-AC addition of 0.5% in Comparative Example 2, the amplification rate was significantly delayed and the Ct value was 25.2. From this result, it can be seen that PCR is inhibited when 0.1 μL of TM-AC is added to the PCR tube (20 μL of reaction solution) (TM-AC final concentration 0.5%).
　ＴＭ－ＡＣとＴｗｅｅｎ２０（終濃度５％）をＰＣＲチューブに同時に添加した結果を図１（黒四角：Ｔｗｅｅｎ２０添加あり）に示す。対照例２のＴＭ－ＡＣ添加０％からわかるように、５％Ｔｗｅｅｎ２０によるＰＣＲ阻害はみられなかった。実施例２の０．５％ＴＭ－ＡＣと５％Ｔｗｅｅｎ２０を同時添加した時のＣｔ値は２１．０であることから、Ｔｗｅｅｎ２０がＰＣＲ阻害を軽減したことがわかる。この結果より、Ｔｗｅｅｎ２０を添加することによって、ＴＭ－ＡＣ　０．１μＬをＰＣＲチューブに直接投入（ＴＭ－ＡＣ終濃度０．５％）し、阻害なくＰＣＲを行うことができることがわかった。 The results of simultaneous addition of TM-AC and Tween 20 (final concentration 5%) to the PCR tube are shown in FIG. 1 (black square: with Tween 20 added). As can be seen from the 0% addition of TM-AC in Control Example 2, PCR inhibition by 5% Tween 20 was not observed. The Ct value when 2% of 0.5% TM-AC and 5% Tween 20 of Example 2 were added simultaneously was 21.0, indicating that Tween 20 reduced PCR inhibition. From this result, it was found that by adding Tween 20, 0.1 μL of TM-AC was directly added to the PCR tube (TM-AC final concentration 0.5%), and PCR could be performed without inhibition.
　本発明は前記実施形態に限定されるものではなく、本発明の目的を達成できる範囲での変形、改良等は本発明に含まれるものである。 The present invention is not limited to the above-described embodiment, but includes modifications and improvements as long as the object of the present invention can be achieved.
　本発明を特定の態様を用いて詳細に説明したが、本発明の意図と範囲を離れることなく様々な変更および変形が可能であることは、当業者にとって明らかである。なお本出願は、２０１５年３月１３日付で出願された日本特許出願（特願２０１５－０５１０７７）に基づいており、その全体が引用により援用される。 Although the present invention has been described in detail using specific embodiments, it will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit and scope of the invention. Note that this application is based on a Japanese patent application filed on March 13, 2015 (Japanese Patent Application No. 2015-051077), which is incorporated by reference in its entirety.
　本発明の遺伝子検査方法によれば、環境・生体試料から標的の微生物、特にクリプトスポリジウムの検出を迅速かつ簡易に行うことができ、且つ、得られた核酸試料を、その後の核酸精製等の煩雑な工程を必要とすることなく、そのまま逆転写反応及び／又は核酸増幅試験に課しても悪影響を与えないため、例えば臨床検査や公衆衛生検査において有用である。 According to the genetic testing method of the present invention, it is possible to quickly and easily detect target microorganisms, particularly Cryptosporidium, from environmental / biological samples, and the obtained nucleic acid sample is subjected to complicated procedures such as subsequent nucleic acid purification. This is useful in clinical examinations and public health examinations, for example, because it does not adversely affect the reverse transcription reaction and / or nucleic acid amplification test as it is without requiring any additional steps.
　１　刺激応答性磁性ナノ粒子
　２　磁性物質
　３　刺激応答性ポリマー
　４　ストレプトアビジン
　５　ビオチン
　６　親和性物質
　７　検出対象 DESCRIPTION OF SYMBOLS 1 Stimulus responsive magnetic nanoparticle 2 Magnetic substance 3 Stimulus responsive polymer 4 Streptavidin 5 Biotin 6 Affinity substance 7 Detection object

Claims (5)

  1. 　被検試料中の核酸を回収し、核酸増幅を行う遺伝子検査方法であって、以下の工程（１）～（４）を含む遺伝子検査方法。
    （１）刺激応答性磁性ナノ粒子及び標的の核酸に対する親和性物質を有する吸着材と前記核酸との結合体を、被検試料を含む水溶液中で生成させる工程
    （２）刺激応答性磁性ナノ粒子が凝集する条件下におき、前記結合体を凝集させる工程
    （３）凝集した結合体を磁石で回収する工程
    （４）回収した結合体を、非イオン性界面活性剤の存在下で核酸増幅を行う工程     A genetic test method for recovering nucleic acid in a test sample and performing nucleic acid amplification, comprising the following steps (1) to (4):
    (1) A step of generating a conjugate of the nucleic acid and the adsorbent having an affinity substance with respect to the target nucleic acid and the nucleic acid in an aqueous solution containing the test sample (2) the stimulus responsive magnetic nanoparticle A step of aggregating the conjugate (3) a step of recovering the aggregated conjugate with a magnet (4) a nucleic acid amplification of the recovered conjugate in the presence of a nonionic surfactant Process to perform
  2. 　前記非イオン性界面活性剤が、ポリオキシエチレン誘導体である請求項１に記載の遺伝子検査方法。 The genetic test method according to claim 1, wherein the nonionic surfactant is a polyoxyethylene derivative.
  3. 　前記ポリオキシエチレン誘導体が、ポリオキシエチレンソルビタンモノラウラート、ポリオキシエチレンソルビタンモノオレアート及びポリオキシエチレンオクチルフェニルエーテルからなる群より選ばれる少なくとも１である請求項２に記載の遺伝子検査方法。 The genetic test method according to claim 2, wherein the polyoxyethylene derivative is at least one selected from the group consisting of polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, and polyoxyethylene octylphenyl ether.
  4. 　前記核酸増幅を行う工程における、前記非イオン性界面活性剤の濃度が、０．１～５質量％である、請求項１～３のいずれか１項に記載の遺伝子検査方法。 The gene testing method according to any one of claims 1 to 3, wherein the concentration of the nonionic surfactant in the nucleic acid amplification step is 0.1 to 5% by mass.
  5. 　前記核酸増幅を行う工程で使用する核酸増幅酵素が、Ｔａｑポリメラーゼ、Ｂｓｔポリメラーゼ、又はＡａｃポリメラーゼのいずれかであることを特徴とする、請求項１～４のいずれか１項に記載の遺伝子検査方法。 The gene testing method according to any one of claims 1 to 4, wherein the nucleic acid amplification enzyme used in the nucleic acid amplification step is Taq polymerase, Bst polymerase, or Aac polymerase. .
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