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WO2016148089A1 - Procédé de détection d'une cible de détection à l'aide de nanoparticules magnétiques sensibles à une stimulation - Google Patents

Procédé de détection d'une cible de détection à l'aide de nanoparticules magnétiques sensibles à une stimulation Download PDF

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Publication number
WO2016148089A1
WO2016148089A1 PCT/JP2016/057867 JP2016057867W WO2016148089A1 WO 2016148089 A1 WO2016148089 A1 WO 2016148089A1 JP 2016057867 W JP2016057867 W JP 2016057867W WO 2016148089 A1 WO2016148089 A1 WO 2016148089A1
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detection target
nucleic acid
conjugate
stimulus
acid amplification
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Japanese (ja)
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貴寛 関川
小毛 謝
大西　徳幸
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JNC Corp
University of Shizuoka
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JNC Corp
University of Shizuoka
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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

Definitions

  • the present invention relates to a method for detecting a detection target using stimulus-responsive magnetic nanoparticles.
  • Cryptosporidium is a protozoan that parasitizes the digestive tract of various animals widely distributed throughout the world.
  • the protozoan oocysts are often caused by diarrhea due to water supply due to their strong chlorine tolerance. Therefore, Cryptosporidium has become the most prominent protozoa in clinical and public health.
  • Cryptosporidium is usually detected 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.
  • Magnetic fine particles used in the immunomagnetic bead method include magnetic fine particles (for example, Dynabeads (registered trademark)) whose particle size is designed to be micro size in order to give magnetic beads magnetic separation ability. Yes.
  • Cryptosporidium nucleic acid extraction is performed by physical treatment such as freezing and thawing or chemical treatment using a surfactant or an enzyme (Non-Patent Documents 3 and 4).
  • Reagents used at this time include those requiring careful handling such as strongly alkaline sodium hydroxide solution and highly corrosive phenol solution.
  • Non-Patent Documents 1 and 2 when the target microorganism is separated from the environment / biological sample using the immunomagnetic bead method using the magnetic fine particles described in Non-Patent Documents 1 and 2, the following work is required. That is, before extracting nucleic acids from microorganisms, it is necessary to dissociate microorganisms and magnetic fine particles by adding a predetermined dissociation solution, and to fix the magnetic fine particles with a magnet, and then collect the microorganism concentrate, which is complicated. It was.
  • Non-Patent Documents 3 and 4 for example, after destroying a cell wall or a cell membrane by treatment with a physical or surfactant, an organic solvent such as water-saturated phenol or chloroform is used. Many steps were required, such as using them to remove, denature proteins, and separate nucleic acids. Therefore, in order to process these reagents contained in the extracted nucleic acid sample, a further purification step is required, and the work is complicated.
  • the present inventors have conducted intensive research to find a method capable of easily detecting a detection target regardless of the type of detection target and without using a carefully handled reagent. That is, according to the present invention, when separating the detection target using the magnetic fine particles, it is not necessary to dissociate the collected magnetic fine particles from the detection target, and the nucleic acid can be extracted from the detection target as it is and obtained. It is an object of the present invention to provide a novel detection target detection method that can directly apply a nucleic acid sample to a reverse transcription reaction and / or a nucleic acid amplification test without requiring complicated steps such as subsequent nucleic acid purification. To do.
  • the present inventors separated a detection target using stimulus-responsive magnetic nanoparticles to which an affinity substance for the detection target is bound, added an anionic surfactant to the separated detection target, and subjected to heat treatment And a method for extracting nucleic acid from the detection target was found.
  • the extracted nucleic acid can be directly subjected to the reverse transcription reaction and / or the nucleic acid amplification test without passing through the step of separating the magnetic fine particles and the detection target, and the detection target can be easily detected.
  • the present invention has the following configuration.
  • a method for detecting a detection target in a test sample comprising the following steps (1) to (5).
  • a step of generating a conjugate of an adsorbent having an affinity substance for a stimulus-responsive magnetic nanoparticle and a detection target and the detection target in an aqueous solution containing a test sample (2) Stimulation-responsive magnetic nanoparticle
  • a step of aggregating the conjugate (3) a step of collecting the aggregated conjugate with a magnet (4) adding an anionic surfactant to the recovered conjugate, heat-treating, 1.
  • Step of extracting nucleic acid from detection target (5) Step of performing nucleic acid amplification using the obtained nucleic acid as a sample 2.
  • the anionic surfactant is at least one anion selected from the group consisting of sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS), sodium dodecylbenzenesulfonate (SDBS), sodium cholate, and N-lauroyl sarcosine.
  • SDS sodium dodecyl sulfate
  • LDS lithium dodecyl sulfate
  • SDBS sodium dodecylbenzenesulfonate
  • sodium cholate sodium cholate
  • N-lauroyl sarcosine sodium cholate
  • nucleic acid is used as a concept including DNA, RNA, and derivatives thereof.
  • final concentration indicates the concentration of an anionic surfactant in the reaction tube at the time of nucleic acid amplification test.
  • the present invention it is possible to easily separate a detection target and extract a nucleic acid regardless of the type of a test sample and without using a carefully handled reagent. Furthermore, the obtained nucleic acid sample can be directly subjected to a reverse transcription reaction and / or a nucleic acid amplification test without requiring complicated steps such as subsequent 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 TM-AC concentration on the PCR after the SET method.
  • FIG. 3 is a graph showing the effect of TM-AC concentration on PCR when the SET method is performed on TM-AC from which oocysts have been collected.
  • the present invention is a method for detecting a detection target in a test sample, and includes a detection target including the following steps (1) to (5).
  • a step of generating a conjugate of an adsorbent having an affinity substance for a stimulus-responsive magnetic nanoparticle and a detection target and the detection target in an aqueous solution containing a test sample (2) Stimulation-responsive magnetic nanoparticle
  • a step of aggregating the conjugate (3) a step of collecting the aggregated conjugate with a magnet (4) adding an anionic surfactant to the recovered conjugate, heat-treating, Step of extracting nucleic acid from detection target (5) Step of performing nucleic acid amplification using the obtained nucleic acid as a sample.
  • bonded_body of the adsorbent which has an affinity substance with respect to a stimulus-responsive magnetic nanoparticle and a detection target, and the said detection target in the aqueous solution containing a test sample The detection method of the detection target of this invention
  • the nucleic acid is extracted from the detection target in the test sample, and the detection target is detected by amplifying the nucleic acid.
  • in order to extract the nucleic acid from the detection target first, there is a step of collecting the detection target in the test sample. This step is a step of generating a conjugate of the adsorbent and the detection target in an aqueous solution as the first stage of recovery of the detection target.
  • Test sample The test sample is not particularly limited as long as it contains a detection target described later.
  • water samples existing in the environment such as raw water, river water, lake water, well water, ground water, sewage, waste water, pool water, park water, or ranch soil, farmland soil, lake soil, river soil, etc.
  • environmental samples such as soil samples present in the environment, biological samples such as stool such as humans, livestock and pets, food samples such as beverage foods, raw vegetables and fruits.
  • the detection target is not particularly limited as long as it can be detected by extracting and amplifying a nucleic acid.
  • organisms such as animals (including humans), plants, microorganisms (including protozoa and protozoa) can be targeted.
  • protozoa that formed oocysts such as Cryptosporidium are applicable as detection targets of the present invention.
  • the adsorbent can be used to recover the detection target.
  • the adsorbent has stimulus-responsive magnetic nanoparticles and an affinity substance for the detection target.
  • stimulus-responsive magnetic nanoparticles are used for recovery of a detection target in an aqueous solution containing a test sample.
  • the nucleic acid can be extracted from the detection target while the binding between the stimulus-responsive magnetic nanoparticle and the detection target is not dissociated.
  • the detection target can be easily detected.
  • 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 heat, light, acid, base, pH, or electricity.
  • thermoresponsive polymer a heat-responsive polymer that can be aggregated and dispersed by temperature change
  • thermoresponsive polymer examples 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”).
  • LCST lower critical solution temperature
  • UCST upper critical solution temperature
  • thermoresponsive magnetic nanoparticles having a thermoresponsive polymer Thermo-Max (manufactured by JNC Corporation) can be suitably used.
  • 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 average particle size of the magnetic fine particles is within the above range, the dispersibility in the liquid becomes good.
  • 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.
  • the affinity substance used in the present invention is not particularly limited as long as it binds to the detection target.
  • antibodies, 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 e.g, 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 carried out by a method known in the art such as graft polymerization (for example, ADV. Polym. Sci., Vol. 4, p111, 1965 and J. Polymer Sci., Part-A, 3, p1031). 1965).
  • the adsorbent / detection target combination described above is obtained by combining the adsorbent and the detection target in an aqueous solution by combining the adsorbent and the detection target in an arbitrary method. This produces a conjugate.
  • the binding between the affinity substance and the detection target means specific binding or adsorption, and is not necessarily a covalent binding, and may be binding utilizing ionic or biochemical affinity (affinity). .
  • Examples include binding between an antigen and an antibody, binding between an enzyme and a substrate, binding between a chelating agent and a metal ion, and specific binding such as avidin-biotin.
  • the mixing operation is not particularly limited as long as the adsorbent and the detection target can be brought into contact with each other in an appropriate buffer. For example, it is sufficient that the test sample including the detection target and the tube provided with the adsorbent are lightly agitated or shaken, and examples thereof include a mixing operation using a commercially available vortex mixer.
  • step (2) A step of aggregating the conjugate under conditions where the stimulus-responsive magnetic nanoparticles are aggregated. This step is a second stage of recovery of the detection target, and the adsorbent obtained in step (1) and the detection target In the conjugate, this is a step of aggregating the conjugate under conditions where the stimulus-responsive polymer in the conjugate is aggregated.
  • 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 above 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 the detection target and the adsorbent are combined in an appropriate 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 becomes a supernatant portion from the inside of the tube. By discharging the liquid, the adsorbent to which the detection target is bound can be separated.
  • the magnetic force of a magnet or the like used to separate the adsorbent to which the detection target is coupled differs 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 adding an anionic surfactant to the recovered conjugate, heat-treating, and extracting the nucleic acid from the detection target This step is performed in the conjugate recovered in the above steps (1) to (3). In this step, nucleic acid is extracted from the detection target.
  • the detection target and the anionic surfactant are brought into contact with each other by adding an anionic surfactant to the conjugate. After bringing the detection target into contact with the anionic surfactant, heat treatment is performed to extract the nucleic acid from the detection target.
  • an anionic surfactant as an extraction reagent, it can be applied to any kind of cells such as bacteria and animal cells.
  • anionic surfactant examples include sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS), sodium dodecylbenzenesulfonate (SDBS), sodium cholate, N-lauroyl sarcosine, and the like. From this viewpoint, sodium dodecyl sulfate (SDS) is preferred. In the present invention, various anionic surfactants may be used alone or in combination of two or more.
  • nucleic acid loss may usually occur during the nucleic acid purification process, and the recovery rate of the nucleic acid after purification is at most about 80%.
  • anionic surfactant is used as an extraction reagent.
  • the method of the present invention can be applied to detection targets derived from all living organisms regardless of whether they are animals, plants, or microorganisms.
  • the detection target in the conjugate and an anionic surfactant are brought into contact as necessary. It is also possible to provide a pretreatment step for decomposing these cell walls before. For example, they can be supplied as a stock or concentrated solution of a liquid (solution) dissolved and sealed together with a reagent such as a buffer solution required for each step.
  • cell wall degrading enzymes for example, cellulase, zymolyase, lysozyme, etc.
  • nucleic acid extraction can be performed by the above extraction method without causing cell wall degrading enzymes to act on these organisms.
  • the step of contacting the anionic surfactant with the detection target in the conjugate and heat-treating can be repeated multiple times until the cell membrane of the detection target is sufficiently destroyed, particularly for samples for nucleic acid amplification tests.
  • the time required for the process varies depending on the type and amount of the detection target.
  • the heat treatment is usually performed at 40 to 100 ° C, preferably 80 to 100 ° C.
  • the nucleic acid can be sufficiently extracted from the detection target by performing a heat treatment usually for 5 to 120 minutes, preferably 5 to 60 minutes.
  • the contact time is about 5 to 30 minutes at 90 ° C., cell membranes derived from animals can be sufficiently destroyed.
  • the concentration of the anionic surfactant only needs to be able to extract the quantity and quality necessary for subjecting the obtained nucleic acid to various analyzes such as nucleic acid amplification described below after extracting the nucleic acid from the detection target.
  • concentration of the anionic surfactant only needs to be able to extract the quantity and quality necessary for subjecting the obtained nucleic acid to various analyzes such as nucleic acid amplification described below after extracting the nucleic acid from the detection target.
  • the higher the treatment concentration of the anionic surfactant during nucleic acid extraction the higher the extraction power.
  • the final concentration of the anionic surfactant is preferably 0.001 to 0.1% by mass, more preferably 0.001 to Dilute to 0.01% by weight.
  • the concentration of the anionic surfactant during the nucleic acid extraction treatment is preferably 0.1 to 10% by mass, More preferably, contact with the detection target at a concentration of 0.1 to 1% by mass is preferable for reliable nucleic acid extraction, and further improves the efficiency of nucleic acid extraction and shortens the time required for extraction. , Improve detection sensitivity and achieve rapid detection.
  • the concentration of the anionic surfactant during the nucleic acid extraction treatment is adjusted by diluting the anionic surfactant with, for example, water, 0.1 to 10% by mass, more preferably 0.1 to 1% by mass. It is possible to extract the nucleic acid from the detection target by introducing the conjugate into the solution and performing contact treatment and heat treatment at 90 ° C. for 15 minutes, for example.
  • step (5) In the reverse transcription reaction or nucleic acid amplification test in step (5) described later, if the concentration of the stimulus-responsive magnetic nanoparticles in the reaction solution is high, the reverse transcription reaction or nucleic acid amplification can be inhibited.
  • an anionic surfactant is added to the detection target and the stimulus-responsive magnetic nanoparticles, and the stimulus response is performed by, for example, heating at 90 ° C. for 15 minutes. Inhibition of reverse transcription reaction and nucleic acid amplification by the magnetic nanoparticles can be reduced.
  • the reagent form of the anionic surfactant is not particularly limited. As long as the concentration of the anionic surfactant is within the above-described concentration range, it may be in a solution state such as an aqueous solution, or may be in a lyophilized state or a dry state. Moreover, when a reagent form is a freeze-dried state or a dry state, you may combine with the solution for melt
  • nucleic acids from detection targets derived from animals include blood free DNA from blood components such as serum and plasma, DNA such as viral DNA, RNA such as blood free messenger RNA and viral RNA. Is mentioned.
  • Cryptosporidium to be detected refers to a microorganism belonging to the genus Cryptosporidium, and specifically, Cryptosporidium parvum species (hereinafter abbreviated as C. parvum), Cryptosporidium. muris species (hereinafter abbreviated as C. muris), Cryptospodium baileyi species (hereinafter abbreviated as C. baileyi), Cryptosporidium serpentis species (hereinafter abbreviated as C. serpentis), Crytosporidium species, hereinafter. C. meleagridis), Cryptospodium felis species (hereinafter referred to as C. meleagridis) Abbreviated as C.Feris.) And the like, the method of the present invention is used to detect these species to species discriminative.
  • C. parvum Cryptosporidium parvum
  • C. muris Cryptosporidium. muris species
  • C. baileyi Cryptospodium baileyi species
  • C. serpentis Cryptosporidium serpentis species
  • nucleic acid extraction from oocysts only requires treatment with an anionic surfactant, and there is no need to perform complicated steps such as protease treatment and purification treatment as in the prior art.
  • the nucleic acid extraction treatment conditions with such an anionic surfactant are preferably, for example, 5 to 30 minutes at 60 to 100 ° C., and particularly preferably 10 to 20 minutes at 90 ° C.
  • the nucleic acid extraction method described above is called a surfactant extraction treatment (hereinafter referred to as SET) method, and is the method described in the 48th Annual Meeting of the Japan Society on Water Environment, p.421 (2014).
  • SET surfactant extraction treatment
  • the concentration of the anionic surfactant used for the nucleic acid extraction treatment is 0.1 to 10% by mass, preferably 0.1 to 1% by mass (the anionic surfactant during nucleic acid amplification). In the case of diluting the sample treated in step 10), it is preferable to adjust. By being in the above range, inhibition of nucleic acid amplification by an anionic surfactant can be suppressed.
  • Step of performing nucleic acid amplification using the obtained nucleic acid as a sample This step is a step of performing nucleic acid amplification using the nucleic acid obtained in the above step as a sample.
  • concentration of stimuli-responsive magnetic nanoparticles in the adsorbent is high, reverse transcription reaction and nucleic acid amplification can be inhibited.
  • the inhibition of reverse transcription reaction and nucleic acid amplification by the stimulus-responsive magnetic nanoparticles is reduced by heat-treating the stimulus-responsive magnetic nanoparticles together with the detection target in step (4) with an anionic surfactant. can do.
  • the stimulus-responsive magnetic nanoparticles having excellent dispersibility are used as the magnetic fine particles, even if the magnetic fine particles remain in the reaction solution used in the reverse transcription reaction or nucleic acid amplification test described below. Since it is appropriately dispersed in the liquid, a reverse transcription reaction and a nucleic acid amplification test can be performed efficiently.
  • the obtained nucleic acid is RNA
  • reverse transcription reaction is performed to obtain DNA, and then nucleic acid amplification is performed.
  • any conventionally known method can be employed using the RNA obtained in the above steps (1) to (4) 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 method, reverse transcription SmartAmp method and the like.
  • 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 method, reverse transcription SmartAmp method and the like.
  • 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.
  • the anionic surfactant remains in the sample and becomes an inhibitor during reverse transcription reaction or nucleic acid amplification. Accordingly, when the nucleic acid extract is subjected to reverse transcription reaction or nucleic acid amplification, the final concentration of the anionic surfactant is preferably 0.0001 to 0.01% by mass, more preferably 0.0001 to 0.00. Dilute to 001% by weight. By being in the above range, inhibition of nucleic acid amplification by an anionic surfactant can be suppressed.
  • the stimulus-responsive magnetic nanoparticles remaining in the sample are preferably 0.1 to 10% by mass, more preferably 0.5 to 1% by mass. By being in the above range, inhibition of nucleic acid amplification by the stimulus-responsive magnetic nanoparticles can be reduced.
  • This step is preferably added after step (5).
  • This step is a step of calibrating the DNA chain fragment (PCR amplification product) amplified in step (4) and determining the presence or absence of the detection target in the test sample.
  • the analysis method in this step is not particularly limited as long as the PCR amplification product can be detected or quantified.
  • electrophoresis method real-time PCR method and the like can be mentioned.
  • the electrophoresis method the amount and size of the PCR amplification product can be evaluated.
  • PCR amplification products can be quickly quantified.
  • the change in fluorescence intensity is generally a noise level and is equal to zero until the number of amplification cycles is 1 to 10. Therefore, these are regarded as sample blanks with zero amplification products, and their standard deviation SD is calculated.
  • the fluorescence value multiplied by 10 is defined as a threshold value, and the number of PCR cycles that first exceeds the threshold value is referred to as a cycle threshold value (Ct value). Therefore, the larger the initial DNA template amount in the PCR reaction solution, the smaller the Ct value, and the smaller the template DNA amount, the larger the Ct value. Even if the amount of template DNA is the same, the Ct value of the PCR reaction in the same region becomes larger as the ratio of cleavage in the PCR target sequence region in the template increases.
  • a primer common to a plurality of types of detection targets when used, a plurality of types of detection targets in a test sample can be detected. Further, when a primer specific to a specific detection target is used, the specific detection target in the test sample can be detected.
  • PCR conditions are not particularly limited as long as specific amplification occurs in accordance with the principle of PCR, and can be set as appropriate.
  • Test Example 1 In Test Example 1, the effect of stimulation-responsive magnetic nanoparticles heat-treated with an SDS solution on real-time PCR was evaluated.
  • 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 to Cryptosporidium 18S rDNA gene forward: 5′-GGAAGGGTTGTATTATTATATAAAGAAACC-3 ′ (SEQ ID NO: 2), reverse: 5′-CTCCCTCCTCGAGAATCGAA-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, real-time PCR (LightCycler Nano, Roche Diagnostics Inc.) was used.
  • 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.
  • Reference Example 1-2 Reference Example 1-2 was tested in the same manner as Reference Example 1-1 except that the SET method for TM-AC was not performed.
  • Reference Example 2-1 and Reference Example 2-2 Reference Example 2-1 was tested in the same manner as Reference Example 1-1 except that the final concentration of TM-AC was adjusted to 0.5%.
  • Reference Example 2-2 was tested in the same manner as Reference Example 1-1 except that the SET method for TM-AC was not performed and the final concentration of TM-AC was adjusted to 0.5%.
  • Reference Example 3-1 and Reference Example 3-2 Reference Example 3-1 was tested in the same manner as Reference Example 1-1 except that the final concentration of TM-AC was adjusted to 1.0%.
  • Reference Example 3-2 was tested in the same manner as Reference Example 1-1 except that the SET method for TM-AC was not performed and the final concentration of TM-AC was adjusted to 1.0%.
  • Control Example 1 Control Example 2
  • Control 1 was tested in the same manner as Reference Example 1-1 except that the final TM-AC concentration was 0%.
  • Control 2 was tested in the same manner as Reference Example 1-1 except that the SET method for TM-AC was not performed and the final concentration of TM-AC was 0%.
  • PCR inhibition by TM-AC can be reduced by PCR using TM-AC after SET. It was also found that PCR can be performed without inhibition even when 0.2 ⁇ L of TM-AC (TM-AC final concentration 1%) is directly added to the PCR tube (20 ⁇ L of reaction solution) after SET.
  • Example 2 In Test Example 2, Cryptosporidium oocysts were collected using TM-AC, the SET method was performed on TM-AC together with the collected oocysts, DNA was extracted from the oocysts, and real-time PCR was performed together with TM-AC. The detection sensitivity was evaluated. [Examples 1 to 3] (1) Recovery of Cryptosporidium oocysts Add 100 ⁇ L of the above TM-AC to oocyst-containing water (10 5 pieces / 750 mL), and incubate at room temperature for 15 minutes while slowly rotating (18 rpm) to bind TM-AC and oocysts. The body was made. Immediately after heating at 42 ° C. for 1 minute, a microtube was set on a magnetic board and placed for 1 minute or longer to magnetically separate TM-AC and oocyst conjugate. After magnetic separation, the supernatant was removed.
  • TM-AC was dispersed by adding 100 ⁇ L of TE buffer (concentration 0.1%) containing TE buffer (pH 8.0, 0.01 M Tris-TCl, 1 mM EDTA-Na 2 ) (approximately the same concentration as the initial TM-AC concentration). To prepare). Thereafter, heat treatment was performed in a water bath (90 ° C., 15 minutes) to perform a SET method (Surfant extraction treatment method (surfactant extraction treatment method)), and DNA was extracted from oocysts.
  • TE buffer concentration 0.1%) containing TE buffer (pH 8.0, 0.01 M Tris-TCl, 1 mM EDTA-Na 2 ) (approximately the same concentration as the initial TM-AC concentration).
  • quantified oocysts (Iowa Isolate, Waterborne) recovered from the stool of mice using a density gradient centrifugation method were used by orally administering Cryptospodium parvum oocysts to mice. It was.
  • PCR was performed using 0.8 ⁇ L primer set specific for Cryptosporidium 18S rDNA gene (5 ⁇ M each) and 2.0 ⁇ L TaqMan probe (1 ⁇ M), 10 ⁇ L PCR reagent Premix EX Taq (Takara Bio Inc.), real-time PCR (LightCycler Nano, Roche) ⁇ Diagnostics Co., Ltd.)
  • 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 was analyzed with LightCycler Nano software.
  • DNA was extracted from the TM-AC from which oocysts were collected by the SET method, and then the DNA detection sensitivity was evaluated. Details of the samples used for PCR are shown in Table 1, and the results of real-time PCR are shown in FIG.
  • the sample (200 oocysts) having a dilution ratio of 1/10 in Example 1 contained 1% TM-AC that inhibits PCR, but the inhibition was suppressed by SET and DNA was detected. Further, DNA could also be detected from the 20 oocyst samples of Example 2 (dilution ratio 1/100) and the 2 oocyst samples of Example 3 (dilution ratio 1/1000).
  • TM-AC can efficiently recover oocysts, and that DNA can be extracted from the mixture of TM-AC and oocysts by the SET method.
  • PCR can be performed without a DNA purification step by directly introducing 0.2 ⁇ L of TM-AC (1% TM-AC final concentration) after SET into a PCR tube (20 ⁇ L of reaction solution).
  • 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.
  • target microorganisms particularly Cryptosporidium
  • the obtained nucleic acid sample can be subjected to complicated nucleic acid purification and the like. It 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 a step.

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Abstract

La présente invention résoud le problème consistant à fournir un procédé permettant de détecter facilement une cible de détection indépendamment de la classification de la cible de détection et sans utiliser de réactif qui nécessite une manipulation prudente. La présente invention concerne un procédé de détection d'une cible de détection qui détecte une cible de détection dans un échantillon d'essai et qui comprend les étapes (1)-(5) suivantes : (1) une étape au cours de laquelle des nanoparticules magnétiques sensibles à une stimulation et un conjugué d'une cible de détection et d'un matériau d'adsorption qui comprend une substance ayant une affinité pour la cible de détection sont générés dans une solution aqueuse qui contient un échantillon d'essai, (2) une étape au cours de laquelle le conjugué est aggloméré dans des conditions où les nanoparticules magnétiques sensibles à une stimulation s'agglomèrent, (3) une étape au cours de laquelle le conjugué aggloméré est collecté au moyen d'un aimant, (4) une étape au cours de laquelle un tensioactif anionique est ajouté au conjugué collecté, un traitement thermique est effectué, et un acide nucléique est extrait de la cible de détection, et (5) une étape au cours de laquelle une amplification de l'acide nucléique est effectuée en utilisant l'acide nucléique acquis en tant qu'échantillon.
PCT/JP2016/057867 2015-03-13 2016-03-11 Procédé de détection d'une cible de détection à l'aide de nanoparticules magnétiques sensibles à une stimulation Ceased WO2016148089A1 (fr)

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