JP6762082B2 - Method of detecting a detection target using stimulus-responsive magnetic nanoparticles - Google Patents

Description

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, which is widely distributed all over the world. Cryptosporidium is currently the most noticeable protozoan clinically or in public health, as oocysts of this protozoan often cause mass diarrhea via water due to their strong chlorine resistance. ..

The detection of Cryptosporidium is usually performed visually under a microscope, but such a method requires a great deal of labor for detection because the operation is complicated, the speed is low, and the detection rate is low. Therefore, in recent years, genetic methods have come to be used for the study of Cryptosporidium, and it has become relatively easy to identify and type species.

Conventionally, the immunomagnetic bead method has been used as a method for separating oocysts from environmental / biological samples (Non-Patent Documents 1 and 2). As the magnetic fine particles used in the immunomagnetic bead method, magnetic fine particles whose particle size is designed to have a micro size (for example, Dynabeads (registered trademark)) are used in order to give the magnetic beads magnetic separation ability. There is.

Further, for example, the nucleic acid extraction of Cryptosporidium is carried out by physical treatment such as freeze-thaw or chemical treatment with a surfactant or an enzyme (Non-Patent Documents 3 and 4). The reagents used at this time include those that require careful handling, such as a strongly alkaline sodium hydroxide solution and a highly corrosive phenol solution.

EPA Method 1623 (US Environmental Protection Agency) Inspection method for indicator bacteria and cryptosporidium in water supply (Notice of Waterworks Division Manager, Health Bureau, Ministry of Health, Labor and Welfare, No. 0302, issued on March 2, 2012) Diagnostic manual for protozoan diarrhea centered on cryptosporidium disease, 2003, National Institute of Infectious Diseases) Applied and Environmental Microbiology, Vol. 61, No. 11 (1995), 3849-3855.

However, when the target microorganism is separated from the environment / biological sample by using the immunomagnetic bead method using the magnetic fine particles described in Non-Patent Documents 1 and 2, a predetermined dissociation solution is used before extracting the nucleic acid from the microorganism. It is necessary to dissociate the microorganisms and the magnetic fine particles by adding the above, fix the magnetic fine particles with a magnet, and then recover the microbial concentrate, which complicates the work.

Further, as a method for extracting the nucleic acid described in Non-Patent Documents 3 and 4, for example, a cell wall or a cell membrane is physically or treated with a surfactant or the like to destroy it, and then an organic solvent such as water-saturated phenol or chloroform is used. Many steps were required, such as removing them using, denaturing proteins, and separating nucleic acids. Therefore, a further purification step is required to process these reagents contained in the extracted nucleic acid sample, which complicates the work.

Therefore, the present invention has found a method capable of easily detecting a detection target regardless of the type of the detection target and without using a reagent with careful handling. That is, when separating the detection target using the magnetic fine particles, it is not necessary to dissociate the recovered magnetic fine particles and the detection target, and the nucleic acid can be extracted from the detection target as it is, and the obtained nucleic acid sample can be obtained. An object of the present invention is to provide a novel detection method for a detection target, which can be directly applied to a reverse transcription reaction and / or a nucleic acid amplification test without requiring a complicated step such as subsequent nucleic acid purification.

The present inventors separated the detection target using stimulus-responsive magnetic nanoparticles to which an affinity substance for the detection target is bound, and added an anionic surfactant to the separated detection target for heat treatment. By performing the above and extracting the nucleic acid from the detection target, the extracted nucleic acid can be directly subjected to the reverse transcription reaction and / or the nucleic acid amplification test without going through the step of separating the magnetic fine particles and the detection target. It was found that the detection target can be detected, and the present invention has been completed.
Specifically, the present invention has the following configuration.

1. 1. A method for detecting a detection target in a test sample, which comprises the following steps (1) to (5).
(1) A step of forming a stimulus-responsive magnetic nanoparticles and a conjugate of the adsorbent having an affinity substance for the detection target and the detection target in an aqueous solution containing a test sample (2) Stimulation-responsive magnetic nanoparticles Steps of aggregating the conjugates (3) Steps of recovering the aggregated conjugates with a magnet (4) An anionic surfactant is added to the collected conjugates and heat-treated. Step of extracting nucleic acid from the detection target (5) Step of performing nucleic acid amplification using the obtained nucleic acid as a sample. The method for detecting a detection target according to 1 above, wherein the final concentration of the anionic surfactant in the step of performing nucleic acid amplification is 0.0001 to 0.01% by mass.
3. 3. The anionic surfactant is at least one anion selected from the group consisting of sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS), sodium dodecylbenzene sulfonate (SDBS), sodium cholate, and N-lauroyl sarcosin. The method for detecting an object to be detected according to 1 or 2 above, which is an ionic surfactant.
4. The method for detecting a detection target according to any one of 1 to 3 above, wherein the concentration of the stimulus-responsive magnetic nanoparticles in the step of performing nucleic acid amplification is 0.1 to 10% by mass.
5. The method for detecting a detection target according to any one of 1 to 4 above, wherein the nucleic acid amplification enzyme used in the step of performing nucleic acid amplification is any one of Taq polymerase, Bst polymerase, and Aac polymerase. ..

In addition, in this specification, "nucleic acid" is used as a concept including DNA, RNA and derivatives thereof.
Further, in the present specification, the "final concentration" indicates the concentration of the anionic surfactant in the reaction tube at the time of the nucleic acid amplification test.

According to the present invention, it is possible to easily separate the detection target and extract the nucleic acid regardless of the type of the test sample and without using a reagent with careful handling, and the obtained nucleic acid sample can be obtained. It can be directly subjected to a reverse transcription reaction and / or a nucleic acid amplification test without requiring a complicated step such as subsequent nucleic acid purification.

FIG. 1 is a schematic configuration diagram of an adsorbent in the method according to the embodiment of the present invention. FIG. 2 is a graph showing the effect of the TM-AC concentration after the SET method on PCR. FIG. 3 is a graph showing the effect of the concentration of TM-AC on PCR when the SET method is performed on TM-AC from which oocysts have been recovered.

Hereinafter, an embodiment of the present invention will be described.
The present invention is a method for detecting a detection target in a test sample, and is a method for detecting a detection target including the following steps (1) to (5).
(1) A step of forming a stimulus-responsive magnetic nanoparticles and a conjugate of the adsorbent having an affinity substance for the detection target and the detection target in an aqueous solution containing a test sample (2) Stimulation-responsive magnetic nanoparticles Steps of aggregating the conjugates (3) Steps of recovering the aggregated conjugates with a magnet (4) An anionic surfactant is added to the collected conjugates and heat-treated. Step of extracting nucleic acid from the detection target (5) Step of performing nucleic acid amplification using the obtained nucleic acid as a sample.

Each step will be described below.
(1) A step of forming a conjugate of a stimulus-responsive magnetic nanoparticle and an adsorbent having an affinity substance for a detection target and the detection target in an aqueous solution containing a test sample. The detection method of the detection target of the present invention is , The detection target is detected by extracting the nucleic acid from the detection target in the test sample and amplifying the nucleic acid. In the present invention, in order to extract nucleic acid from a detection target, first, there is a step of recovering the detection target in the test sample. This step is a step of forming an adsorbent and a conjugate to be detected in an aqueous solution as the first step of collecting the detection target.

(Test sample)
The test sample is not particularly limited as long as it contains a detection target, but for example, the environment such as tap water, river water, lake water, well water, groundwater, sewage, waste water, pool water, and park water. Water samples existing in, environmental samples such as soil samples existing in the environment such as ranch soil, farmland soil, lake soil, river soil, biological samples such as feces of humans, livestock, pets, beverage foods, etc. Examples thereof include food samples such as raw vegetables and fruits.

(Detection target)
The detection target is not particularly limited as long as it can be detected by extracting and amplifying nucleic acid, and for example, animals (including humans), plants, microorganisms (including protozoans, protozoa, etc.) and the like. It is possible to target living things. As the detection target, for example, even a protozoan forming an oocyst such as Cryptosporidium can be applied as a detection target of the present invention.

(Adsorbent)
In 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)
In the present invention, stimulus-responsive magnetic nanoparticles are used to recover the detection target in the aqueous solution containing the test sample. By using the stimulus-responsive magnetic nanoparticles, the nucleic acid is extracted from the detection target while the stimulus-responsive magnetic nanoparticles and the detection target are not dissociated, and the subsequent nucleic acid amplification test or the like is performed. It can be carried out, and the detection target can be easily detected.

The stimulus-responsive magnetic nanoparticles used in the present invention are obtained by using a stimulus-responsive polymer and a magnetic substance (magnetic fine particles). This stimulus-responsive polymer is a magnetic nanoparticle that can undergo structural changes in response to external stimuli to regulate aggregation and dispersion. The stimulus may be various physical or chemical signals such as heat, light, acid, base, pH or electricity, but not particularly limited.

In particular, as the stimulus-responsive polymer, a heat-responsive polymer that can aggregate and disperse by changing the temperature can be used. Examples of the heat-responsive polymer include a polymer having a lower limit critical solution temperature (hereinafter, also referred to as LCST) and a polymer having an upper limit critical solution temperature (hereinafter, also referred to as “UCST”). As the heat-responsive magnetic nanoparticles having a heat-responsive polymer, Therma-Max (manufactured by JNC Corporation) can be preferably used.

Examples of the polymer having the lower limit critical solution temperature used in the present invention include N-n-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, and N-acryloylpiperidin. , N-acrylloylmorpholin, Nn-propylmethacrylate, N-isopropylmethacrylate, N-ethylmethacrylate, N, N-dimethylmethacrylate, N-methacryloylpyrrolidine, N-methacryloylpiperidin, N-methacryloylmorpholine, etc. Polymers consisting of N-substituted (meth) acrylamide derivatives; polyoxyethylene alkylamine derivatives such as hydroxypropyl cellulose, polyvinyl alcohol partial vinegar, polyvinyl methyl ether, (polyoxyethylene-polyoxypropylene) block copolymer, polyoxyethylene laurylamine, etc. Polyoxyethylene sorbitan ester derivatives such as polyoxyethylene sorbitan laurate; (polyoxyethylene alkylphenyl ether) (meth) acrylates such as (polyoxyethylene nonylphenyl ether) acrylates and (polyoxyethylene octylphenyl ether) methacrylates ; And polyoxyethylene (meth) acrylic acid ester derivatives such as (polyoxyethylene alkyl ether) (meth) acrylates such as (polyoxyethylene lauryl ether) acrylate and (polyoxyethylene oleyl ether) methacrylate. Further, for example, a copolymer consisting of these polymers and at least two kinds of these monomers can be mentioned. Further, for example, a copolymer of N-isopropylacrylamide and N-t-butylacrylamide can be mentioned.

When a polymer containing a (meth) acrylamide derivative is used, other copolymerizable monomers may be copolymerized with the polymer in a range having a lower critical solution temperature. In the present invention, among them, N-n-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acrylloylpyrrolidin, N-acrylloylpiperidin, N-acrylloylmorpholin, Nn- From at least one monomer selected from the group consisting of propyl methacrylamide, N-isopropyl methacrylamide, N-ethyl methacrylamide, N, N-dimethyl methacrylamide, N-methacryl pyrrolidin, N-methacryl piperidin, N-methacryl morpholine. Polymers or copolymers of N-isopropylacrylamide and N-t-butylacrylamide are preferably available.

The polymer having an upper critical solution temperature used in the present invention is, for example, a polymer composed of at least one monomer selected from the group consisting of acroyl glycinamide, acroyl nipecotamide, acryloyl asparagine amide, acryloyl glutamine amide and the like. Can be mentioned. Further, it may be a copolymer composed of at least two of these monomers. These polymers include other copolymers such as acrylamide, acetylacrylamide, biotinol acrylate, N-biotinyl-N'-methacloyl trimethyleneamide, acroylzarcosinamide, methacrylzarcosinamide, acroylmethyluracil, etc. Possible monomers may be copolymerized in a range having an upper critical solution temperature.

Further, in the present invention, as a stimulus-responsive polymer, a pH-responsive polymer that can aggregate and disperse by changing pH can be used.
Examples of such a pH-responsive polymer include polymers 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 salts thereof as copolymerization components.

The magnetic fine particles, which are magnetic substances, are composed of, for example, a 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 as a constituent unit and capable of binding to iron ions, and examples thereof include dextran, polyvinyl alcohol, mannitol, sorbitol and cyclodextrin. For example, Japanese Patent Application Laid-Open No. 2005-82538 discloses a method for producing a fine particle magnetic substance using dextran. Further, a compound having an epoxy such as a glycidyl methacrylate polymer and forming a polyhydric alcohol structure after ring opening can also be used.

The fine particle-like magnetic substance (magnetic fine particles) prepared by 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 order to increase the detection sensitivity of the target detection target. Further, when the average particle size of the magnetic fine particles is within the above range, the dispersibility in the liquid is improved, and as a result, the magnetic fine particles do not settle in the reaction liquid during the nucleic acid amplification test and are appropriately dispersed, resulting in efficiency. You can often perform reverse transcription reactions and nucleic acid amplification tests.

(Affinity substance)
The affinity substance used in the present invention is not particularly limited as long as it binds to the detection target, and is, for example, an antibody, an aptamer, or a substance having a cationic functional group (for example, a primary amino group). Substances with secondary amino groups, tertiary amino groups, quaternary ammonium groups or guanidino groups), substances with anionic functional groups, proteins that interact with target substances (eg, enzymes, receptors), chelating agents, etc. Can be mentioned.

(Bound of stimulus-responsive magnetic nanoparticles and affinity substances)
The method of binding the stimulus-responsive magnetic nanoparticles to the affinity substance is not particularly limited, but for example, the stimulus-responsive magnetic nanoparticles side (for example, the stimulus-responsive polymer portion) and the affinity substance side are compatible with each other. Substances (eg, biotin and streptavidin or avidin) are bound, and stimulus-responsive magnetic nanoparticles and affinity substances are bound via these substances.

Specifically, the binding of biotin to a stimulus-responsive polymer is addition-polymerizable by binding biotin or the like with a polymerizable functional group such as methacryl or acrylic, as described in International Publication No. 01/09141. This is performed by using a monomer and copolymerizing it with another monomer, and streptavidin is immobilized therein. In addition, labeling of the affinity substance with biotin or the like is carried out according to a conventional method. When the biotin-labeled affinity substance 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.

Specifically, as shown in FIG. 1, the stimulus-responsive magnetic nanoparticles 1 contain fine-grained magnetic substance 2, and the 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 the affinity substance 6 for the detection target 7 is bound via biotin 5.

In the present specification, a material in which a stimulus-responsive polymer and a fine particle-like magnetic substance are bonded is referred to as stimulus-responsive magnetic nanoparticles, and when the stimulus is thermal, it is referred to as heat-responsive magnetic nanoparticles. There is.

The bond between the finely divided magnetic substance and the stimulus-responsive polymer can be obtained by a method of bonding via a reactive functional group or by introducing a polymerizable unsaturated bond into the active hydrogen or polyvalent alcohol on the polyvalent alcohol in the magnetic substance. Then, it may be carried out by a method well known in the art such as a method of graft polymerization (for example, ADV. Polymer. Sci., Vol. 4, p111, 1965, J. Polymer Sci., Part-A, 3, p1031). , 1965).

(Formation of adsorbent and the conjugate to be detected in an aqueous solution)
In the above-mentioned conjugate of the adsorbent and the detection target, the adsorbent and the detection target are mixed in an aqueous solution by an arbitrary method, so that the affinity substance in the adsorbent and the detection target are combined, and the adsorbent and the detection target are combined. Is produced. The bond between the affinity substance and the detection target means a specific bond or adsorption, and does not necessarily have to be a covalent bond, but may be a bond using ionic or biochemical affinity (affinity). .. For example, the binding between an antigen and an antibody, the binding between an enzyme and a substrate, the binding between a chelating agent and a metal ion, and a specific binding such as avidin-biotin can be exemplified. The mixing operation is not limited as long as the adsorbent can come into contact with the detection target in a suitable buffer. For example, it is sufficient to lightly invert or shake the test sample including the detection target 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) A step of aggregating the conjugate under conditions where stimulus-responsive magnetic nanoparticles are agglomerated This step is the second step of collecting the detection target, and the adsorbent obtained in the step (1) and the detection target This is a step of aggregating the conjugate under the condition that the stimulus-responsive polymer in the conjugate aggregates in the conjugate.

To set the conditions for the stimulus-responsive polymer to aggregate, for example, when a heat-responsive polymer is used, the container containing the mixture may be moved to a constant temperature bath at a temperature at which the heat-responsive polymer aggregates. There are two types of heat-responsive polymers, a polymer having an upper critical solution temperature and a polymer having a lower critical solution temperature. For example, when a polymer having a lower critical solution temperature of 37 ° C. is used, the heat-responsive polymer can be aggregated by moving the container containing the mixture to a constant temperature bath of 37 ° C. or higher. Further, when a polymer having an upper critical solution temperature of UCST of 5 ° C. is used, the heat-responsive polymer can be aggregated by moving the container containing the mixture to a constant temperature bath of less than 5 ° C.

Here, the lower limit critical solution temperature and the upper limit critical solution temperature are determined as follows, for example. First, the sample is placed in the cell of the absorptiometer and the temperature of the sample is raised 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 the transmittance when completely aggregated is 0%, the temperature at which the transmittance becomes 50% is determined as LCST. In the case of UCST, the sample is cooled at a rate of 1 ° C./min, and the following is obtained by the same method as LCST.

When a pH-responsive polymer is used, an acid solution or an alkaline solution may be added to the 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 in which the pH-responsive polymer causes a structural change, and the pH-responsive polymer changes the structure in the container. The pH range may be changed to raise. For example, when a pH-responsive polymer that aggregates at pH 5 or lower and disperses at pH 5 or higher is used, an acid solution may be added to a container containing the mixture dispersed at pH 5 or higher so that the pH becomes 5 or lower. When a pH-responsive polymer that aggregates at pH 10 or higher and disperses at pH 10 or higher is used, an alkaline solution may be added to a container containing the mixture dispersed at pH 10 or higher so that the pH becomes 10 or higher. The pH at which the pH-responsive polymer causes a structural change is not particularly limited, but is preferably pH 4 to 10, and more preferably pH 5 to 9.

When a photoresponsive polymer is used, the container containing the mixture may be irradiated with light having a wavelength at which the polymer can be aggregated. The preferred light for agglomeration varies depending on the type and structure of the photoresponsive functional groups contained in the photoresponsive polymer, but in general, ultraviolet light or visible light having a wavelength of 190 to 800 nm can be preferably used. At this time, the strength is preferably 0.1 to 1000 mW / cm ² . The photoresponsive polymer is preferably one that does not easily disperse when irradiated with light used for measuring turbidity, in other words, one that aggregates, in that the measurement accuracy can be improved. When a photoresponsive polymer that causes dispersion when irradiated with light used for measuring turbidity is used, the measurement accuracy can be improved by shortening the irradiation time.

(3) Step of recovering the aggregated conjugate with a magnet This step is a step of separating the aggregated conjugate in step (2) with a magnet and recovering the detection target as the final step of collecting the detection target. By applying a magnetic force to the conjugate containing the 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.

For example, when the detection target and the adsorbent are bonded 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 the supernatant portion is formed 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 bound differs depending on the magnitude of the magnetic force of the magnetic substance used. As the magnetic force, a magnetic force capable of concentrating the target magnetic substance can be appropriately used. As the material of the magnet, for example, a material made of the above-mentioned magnetic substance material can be used. For example, a neodymium magnet (manufactured by 26 Seisakusho Co., Ltd.) can be used. The magnetic force of the neodymium magnet is more preferably 3800 gauss or more.

(4) Step of adding an anionic surfactant to the recovered conjugate, heat-treating, and extracting nucleic acid from the detection target This step is a step in the conjugate recovered in the above steps (1) to (3). This is a step of extracting nucleic acid from the detection target. In the present invention, in order to extract nucleic acid from the detection target in the recovered conjugate, the detection target and the anionic surfactant are brought into contact with each other by adding an anionic surfactant to the conjugate. After contacting the detection target with the anionic surfactant, heat treatment is performed to extract nucleic acid from the detection target. By using an anionic surfactant as an extraction reagent, it can be applied to any kind of cells such as bacteria and animal cells.

Examples of the anionic surfactant include sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS), sodium dodecylbenzene sulfonate (SDBS), sodium colate, N-lauroyl sarcosin and the like, and have a nucleic acid extraction ability. From this point of view, sodium dodecyl sulfate (SDS) is preferable. In the present invention, various anionic surfactants may be used alone or in combination of two or more.

Further, by using an anionic surfactant as the extraction reagent, an extraction reagent other than the anionic surfactant is not required, so that the nucleic acid can be extracted from the detection target easily and at low cost. Further, usually, nucleic acid loss may occur in the process of nucleic acid purification, and the recovery rate of nucleic acid after purification is at most about 80%, but only an anionic surfactant is used as an extraction reagent. When the extraction reaction is carried out, the loss of nucleic acid due to nucleic acid purification can be minimized, so that the nucleic acid extraction efficiency can be maintained at almost 100%, and the detection efficiency can be improved.

In general, since cell membranes are composed of phospholipids and proteins, it is thought that anionic surfactants enter the lipid bilayer of phospholipids in this cell membrane and make the cell membrane fragile, and therefore animals, plants, and microorganisms. The method of the present invention can be applied to detection targets derived from all living organisms regardless of the above.

When extracting nucleic acid from organisms having a cell wall such as plant cells, yeasts, fungi, and Gram-positive bacteria, the detection target in the conjugate is brought into contact with an anionic surfactant, if necessary. It is also possible to provide a pretreatment step to decompose these cell walls in advance. For example, they can be supplied as a stock solution or a concentrated solution of a liquid (solution) dissolved and sealed together with a reagent such as a buffer solution required for each step.

Since the composition of the cell wall differs depending on the species, a cell wall degrading enzyme (for example, cellulase, zymolyase, lysozyme, etc.) corresponding to each composition is brought into contact with the detection target to dissolve the cell wall. However, nucleic acid extraction can be performed by the above extraction method without allowing the cell wall-degrading enzyme to act on these organisms.

The steps of contacting the anionic surfactant with the detection target in the conjugate and heat-treating can be repeated a plurality of times until the cell membrane of the detection target is sufficiently destroyed, and in particular, a sample for a nucleic acid amplification test. In the preparation of a small amount of sample such as the preparation of the above, the time required for the step differs depending on the type and amount of the detection target. The heat treatment is usually carried out at 40 to 100 ° C., preferably 80 to 100 ° C. At this time, when a water bath is used, the nucleic acid can be sufficiently extracted from the detection target by heat-treating for usually 5 to 120 minutes, preferably 5 to 60 minutes. For example, a contact time of about 5 to 30 minutes at 90 ° C. is sufficient to destroy animal-derived cell membranes.

The concentration of the anionic surfactant should be such that after extracting the nucleic acid from the detection target, the amount and quality required for subjecting the obtained nucleic acid to various analyzes such as nucleic acid amplification described later can be extracted, and at the time of nucleic acid extraction. The higher the treatment concentration of the anionic surfactant, the higher the extraction power.

When the nucleic acid extract is used for nucleic acid amplification in the step (5) described later, the final concentration of the anionic surfactant is preferably 0.001 to 0.1% by mass, more preferably 0.001 to 0.001. Dilute to 0.01% by weight. Further, in the nucleic acid amplification test, the liquid obtained by the nucleic acid extraction treatment is diluted 10 to 100 times, so that 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 in order to reliably perform nucleic acid extraction, further improving the nucleic acid extraction efficiency and shortening the time required for extraction. , Improving detection sensitivity and achieving rapid detection.

To adjust the concentration of the anionic surfactant during the nucleic acid extraction treatment, the anionic surfactant is diluted with, for example, water, and the above 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 adding the above-mentioned conjugate to the solution and performing contact treatment and heat treatment at 90 ° C. for 15 minutes, for example.

In the reverse transcription reaction and nucleic acid amplification test in the step (5) described later, if the concentration of the stimulus-responsive magnetic nanoparticles in the reaction solution is high, the reverse transcription reaction and nucleic acid amplification can be inhibited. However, as described above, in the nucleic acid extraction step, the stimulus response is obtained by adding an anionic surfactant to the detection target and the stimulus-responsive magnetic nanoparticles and heat-treating at 90 ° C. for 15 minutes, for example. It is possible to reduce the inhibition of reverse transcription reaction and nucleic acid amplification by sexual magnetic nanoparticles.

By bringing the stimulus-responsive magnetic nanoparticles into contact with the anionic surfactant together with the detection target and heat-treating, the reverse transcription reaction and the inhibition of the nucleic acid amplification test by the stimulus-responsive magnetic nanoparticles can be reduced. Although it is not clear, it is presumed that some nucleic acid amplification inhibitor present on the surface of the stimulus-responsive magnetic nanoparticles is destroyed by the above treatment.

The anionic surfactant may be in a solution state such as an aqueous solution containing an anionic surfactant so that the concentration at the time of use is selected from the above-mentioned concentration range, or is selected from the concentration range. It may be in a freeze-dried state or a dried state containing an anionic surfactant so as to have an amount that is sufficient, and the reagent form is not particularly limited.
When the reagent form is in a freeze-dried state or a dry state, it may be combined with a solution for dissolving it, if necessary.

Among the obtained nucleic acids, for example, as nucleic acids from animal-derived detection targets, 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. Can be mentioned.

As a preferred application example of the present invention, this step will be described below using Cryptosporidium, which is one of the microorganisms to be detected.
Here, the Cryptosporidium to be detected refers to a microorganism belonging to the genus Cryptosporidium, and specifically, Cryptosporidium species (hereinafter, abbreviated as C. paravum), Cryptosporidium muris species (hereinafter abbreviated as C. muris), Cryptosporidium baileyi species (hereinafter abbreviated as C. baileyi), Cryptosporidium serpentis species (hereinafter abbreviated as C. serpentis), Cryptosporidium species (hereinafter abbreviated as C. serpentis), Cryptosporidium species C. meleagridis), Cryptosporidium feris species (hereinafter abbreviated as C. feris) and the like are included, and the method of the present invention is used for detecting these species in a species-specific manner.

Extraction of nucleic acid from oocysts only needs to be treated with an anionic surfactant as described above, and it is not necessary to carry out complicated steps such as protease treatment and purification treatment as in the conventional case. The nucleic acid extraction treatment conditions with such an anionic surfactant are preferably, for example, 60 to 100 ° C. for 5 to 30 minutes, and particularly preferably 90 ° C. for 10 to 20 minutes.

The above-mentioned nucleic acid extraction method is called a surfactant extraction treatment (SET) method, and is the method described in "48th Annual Meeting of the Japan Society on Water Environment, p.421 (2014)". ..

As described above, the concentration of the anionic surfactant used in the nucleic acid extraction treatment is 0.1 to 10% by mass, preferably 0.1 to 1% by mass (anionic surfactant during nucleic acid amplification). It is preferable to adjust to (when diluting the sample treated with 10 times). This is because the above range can suppress the inhibition of nucleic acid amplification by the anionic surfactant.

(5) Step of 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. As described above, the stimulus-responsive magnetic nanoparticles in the adsorbent can inhibit the reverse transcription reaction and nucleic acid amplification at high concentrations. However, in the present invention, by heat-treating the stimulus-responsive magnetic nanoparticles together with the detection target with an anionic surfactant in the step (4), the inhibition of the reverse transcription reaction and nucleic acid amplification by the stimulus-responsive magnetic nanoparticles is reduced. can do. Further, in the present invention, since 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 for the reverse transcription reaction and the nucleic acid amplification test described below. Since it is appropriately dispersed in the liquid, the reverse transcription reaction and the nucleic acid amplification test can be efficiently performed.

When the obtained nucleic acid is RNA, reverse transcription reaction is performed to obtain DNA, and then nucleic acid amplification is performed. For the reverse transcription reaction, any conventionally known method can be adopted using the RNA obtained in the above steps (1) to (4) as a template.

Any method can be used as the nucleic acid amplification method, for example, polymerase chain reaction (PCR method), LAMP method, chain substitution amplification, reverse transcriptase chain substitution amplification, reverse transcriptase polymerase chain reaction, reversal. Examples include the copy LAMP method, nucleic acid sequence-based amplification, transcription-mediated amplification and rolling circle amplification method, SmartAmp method, and reverse transcription SmartAmp method.

Usually, DNA amplification is generally performed by the PCR method. As the PCR method, any conventionally known PCR method can be adopted using the DNA obtained in the above step or the DNA obtained by the reverse transcription reaction of RNA as a template. In the present invention, it is particularly preferably available when the nucleic acid amplification enzyme at the time of PCR amplification is any one of Taq polymerase, Bst polymerase, or Aac polymerase.

As described above, the anionic surfactant remains in the sample and becomes an inhibitor during the reverse transcription reaction and nucleic acid amplification. Therefore, when the nucleic acid extract is subjected to a 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. Dilute to 001 mass%. This is because the above range can suppress the inhibition of nucleic acid amplification by the anionic surfactant.

Further, in order to preferably perform the reverse transcription reaction and nucleic acid amplification, 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. Within the above range, inhibition of nucleic acid amplification by stimulus-responsive magnetic nanoparticles can be reduced.

(Step of weighing the amplified DNA fragment to determine the presence or absence of a detection target in the test sample)
This step is preferably further added after step (5). In this step, the DNA strand fragment (PCR amplification product) amplified in step (4) is calibrated to determine the presence or absence of a detection target in the test sample.

The analysis method in this step is not particularly limited as long as it can detect or quantify the PCR amplification product, and examples thereof include electrophoresis method and real-time PCR method. According to the electrophoresis method, the amount of PCR amplification product and its size can be evaluated. Moreover, according to the real-time PCR method, the PCR amplification product can be rapidly quantified. When the real-time PCR method is adopted, the change in fluorescence intensity is generally a noise level and equal to zero from 1 to 10 amplification cycles, so they are regarded as sample blanks with zero amplification product, and their standard deviation SD is calculated. The fluorescence value multiplied by 10 is called the threshold value, and the number of PCR cycles that first exceeds the threshold value is called the cycle threshold value (Ct value). Therefore, the larger the initial amount of DNA template in the PCR reaction solution, the smaller the Ct value, and the smaller the amount of template DNA, the larger the Ct value. Further, even if the amount of template DNA is the same, the greater the proportion of cleavage in the PCR target sequence region in the template, the larger the Ct value of the PCR reaction in the same region.

Further, by using a primer common to a plurality of types of detection targets, it is possible to detect a plurality of types of detection targets in the test sample. Further, if a primer specific to a specific detection target is used, a specific detection target in the test sample can be detected.

The PCR conditions are not particularly limited as long as specific amplification based on the PCR principle occurs, and can be set as appropriate.

When viable bacteria are detected by the methods of the present invention, analysis of PCR amplification products can be performed using standard curves showing the association between microbial abundance and amplification products prepared using standard samples of identified microorganisms. The presence or absence of bacteria or the accuracy of quantification can be improved. Although a standard curve prepared in advance can be used, it is preferable to use a standard curve prepared by performing each step of the present invention on the standard sample at the same time as the test sample. Further, if the correlation between the amount of microorganism and the amount of RNA is investigated in advance, DNA isolated from the microorganism can be used as a standard sample.

The present invention will be described with reference to the following examples, but the present invention is not limited thereto.
However, "%" indicates "mass%".

<Test Example 1>
In Test Example 1, the effect of the stimulus-responsive magnetic nanoparticles heat-treated with the SDS solution on real-time PCR was evaluated.

[Reference Example 1-1]
(1) Preparation of Magnetic Nanoparticles for Oocyst Recovery (TM-AC) 1 ml of heat-responsive magnetic nanoparticles (Therma-Max LA Avidin, 4 g / l, JNC Corporation) to which avidin is bound and C.I. 20 μl of biotinylated monoclonal antibody (Crypt-a-Glo biotin reagent kit (20-fold concentrated), Waterborne) specific for oocysts is mixed and bound, and magnetic nanoparticles for oocyst recovery (Therma-Max-Anti-Cryptosporidium) are mixed and bound. : TM-AC) was produced.

(2) Implementation of SET method (surfactant extraction treatment method) for TM-AC TM-AC and SDS (final concentration 0.1%) prepared in (1) are put into a microtube and heat-treated in a water bath. By (90 ° C., 15 minutes), the SET method (Surfactant extension treatment method (surfactant extraction treatment method)) was carried out on TM-AC.

(3) 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 conditions>
PCR is a primer set specific for the Cryptosporidium 18S rDNA gene
forward: 5'-GGAAGGGTTGTATTATAGATAAAGAACC-3'(SEQ ID NO: 2), reverse: 5'-CTCCCTCTCCGGAATCGAA-3 (SEQ ID NO: 3) (5 μM each) 0.8 μl and TaqMan probe: TCTGACCZATCAGCTT (SEQ ID NO: 4) 0 μl, PCR reagent Premix EX Taq (Takara Bio Inc.) 10 μl, real-time PCR (Light Cycler Nano, Roche Diagnostics Co., Ltd.) was used. The amount of the positive control (5 × 10 ⁶ copies / ml) added was 2 μl, and the amount of the PCR reaction solution was 20 μl.
The 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 (Thrhold Cycle) was analyzed by the Light Cycler 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 Examples 1 and 2]
Control Example 1 was tested in the same manner as Reference Example 1-1, except that the final concentration of TM-AC was set to 0%. Control Example 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 set to 0%.

As reference examples 1-1, 2-1 and 3-1 the evaluation results of the effect of TM-AC after SET on real-time PCR (black square: with SET), and reference examples 1-2, 2-2, 3- As No. 2, the result of TM-AC without SET (white square: without SET) is shown in FIG. The Ct value on the vertical axis represents the number of cycles when the PCR amplification product reaches a certain amount. The larger the amount of PCR amplification product, the smaller the Ct value, and if it is not amplified, it will not be detected.

As can be seen from the addition of TM-AC 0.1% after SET in Reference Example 1-1, PCR inhibition by SET was not observed. In addition, since the Ct value of 0.5% TM-AC after SET in Reference Example 2-1 was 21.8, it was found that PCR inhibition by 0.5% TM-AC was suppressed by SET. .. Furthermore, comparing Reference Example 3-1 and Reference Example 3-2, PCR inhibition of 1% TM-AC was also suppressed by SET, and the Ct value was the same as 0.5% TM-AC.

From this result, it was shown that PCR using TM-AC after SET can reduce PCR inhibition by TM-AC. It was also found that even if 0.2 μl (TM-AC final concentration 1%) of TM-AC was directly added to a PCR tube (20 μl of reaction solution) after SET, PCR could be performed without inhibition.

<Test Example 2>
In Test Example 2, Cryptosporidium oocysts were recovered using TM-AC, the SET method was performed on TM-AC together with the recovered oocysts, DNA was extracted from the oocysts, and then the real-time PCR method was performed together with TM-AC to perform the oocysts. The detection sensitivity was evaluated.
[Examples 1 to 3]
(1) Cryptosporidium was added oocysts above TM-AC 100 [mu] l in the recovery oocyst-containing water (10 ⁵ cells / 750 ml) of, and incubated for 15 minutes at room temperature with slow rotation (18 rpm) binding of TM-AC and oocysts The body was made. Immediately after heating at 42 ° C. for 1 minute, a microtube was set on a magnet plate and left for 1 minute or longer to magnetically separate TM-AC and an oocyst conjugate. After magnetic separation, the supernatant was removed.

(2) Implementation of SET method (surfactant extraction treatment method) for the recovered TM-AC and ocyst conjugate SDS (final) in which the microtube containing the recovered TM-AC and ocyst conjugate is cooled in an ice bath. 100 μl of TE buffer (pH 8.0, 0.01M Tris-TCl, 1 mM EDTA-Na ₂ ) containing (concentration 0.1%) was added to disperse TM-AC (to almost the same concentration as the initial TM-AC concentration). To prepare). Then, the DNA was extracted from the oocyst by performing a SET method (Surfactant extraction treatment method) by heat treatment (90 ° C., 15 minutes) in a water bath.

(3) Implementation of real-time PCR By diluting the DNA extract (SDS 0.1%, TM-AC 100% (almost the same as the product concentration), 1000 ocysts / μl) obtained by the above SET method 10 to 1000 times. The PCR reaction solution was adjusted to the concentration shown in Table 1 and real-time PCR was performed.
<DNA sample>
As the Cryptosporidium DNA sample, DNA extracted from the above oocyst was used. As a positive control, as a positive control of oocysts, a quantified oocyst (Iowa Isolate, Waterborne) obtained by orally administering Cryptosporidium parvum oocysts to mice to infect them and recovering them from the feces of mice by density gradient centrifugation was used. There was.

<PCR conditions>
PCR is 0.8 μl of primer set (5 μM each) and 2.0 μl of TaqMan probe (1 μM) specific for cryptospolidium 18S rDNA gene, 10 μl of PCR reagent Premix EX Taq (Takara Bio Co., Ltd.), real-time PCR (Light Cycler Nano, Roche).・ Diagnostics Co., Ltd.) was used. The amount of the positive control (5 × 10 ⁶ copies / ml) added was 2 μl, and the amount of the PCR reaction solution was 20 μl.
The 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 (Thrhold Cycle) was analyzed by the Light Cycler Nano software.

After extracting DNA from TM-AC from which oocysts were collected by the SET method, the detection sensitivity of DNA was evaluated. The details of the sample used for PCR are shown in Table 1, and the result of real-time PCR is shown in FIG.
The sample with a dilution ratio of 1/10 of Example 1 (200 oocysts) contained 1% TM-AC that inhibits PCR, but the inhibition was suppressed by SET and DNA was detected. In addition, DNA could be detected from 20 samples of oocysts of Example 2 (dilution rate 1/100) and 2 samples of oocysts of Example 3 (dilution rate 1/1000). The Ct value increased as the number of oocysts decreased, and there was a correlation between the Ct value of each dilution and the number of oocysts.
From this result, it was shown that oocysts can be efficiently recovered by TM-AC, and that DNA can be extracted from a mixture of TM-AC and oocysts by the SET method. It was also found that 0.2 μl of TM-AC (TM-AC final concentration 1%) was directly put into a PCR tube (20 μl of reaction solution) after SET, and PCR could be performed without a DNA purification step.

The present invention is not limited to the above-described embodiment, and modifications, improvements, and the like within the range in which the object of the present invention can be achieved are included in the present invention.

1 Stimulus-responsive magnetic nanoparticles 2 Magnetic substances 3 Stimulus-responsive polymers 4 Streptavidin 5 Biotin 6 Affinity substances 7 Detection target

Claims (3)

A method for detecting a detection target in a test sample, which comprises the following steps (1) to (5).
(1) Step of forming a bond between the heat- responsive magnetic nanoparticles and an adsorbent having an affinity substance for the detection target and the detection target in an aqueous solution containing a test sample (2) Heat- responsive magnetic nanoparticles Step of aggregating the conjugates (3) Step of recovering the aggregated conjugates with a magnet (4) Add sodium dodecylsulfate (SDS) to the collected conjugates and heat-treat them. Step of extracting nucleic acid from the detection target (5) Step of performing nucleic acid amplification using the obtained nucleic acid as a sample (however, the concentration of the heat- responsive magnetic nanoparticles in the step of performing the nucleic acid amplification (5) is 0. 5 to 1 % by mass.) The detection method for a detection target according to claim 1, wherein the final concentration of the sodium dodecyl sulfate (SDS) in the step of performing nucleic acid amplification is 0.0001 to 0.01% by mass. The method for detecting a detection target according to claim 1 or 2 , wherein the nucleic acid amplification enzyme used in the step of performing nucleic acid amplification is any one of Taq polymerase, Bst polymerase, and Aac polymerase.

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