JPH11262387A - Nucleic acid-binding magnetic carrier and isolation of nucleic acid using the carrier - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject carrier having a specific outer surface area and capable of isolating nucleic acid from blood in high efficiency by conjugat ing a silica-coated superparamagnetic metal oxide with an inorganic porous wall material composed of fine silica particles. SOLUTION: The objective nucleic acid-binding magnetic carrier containing 10-60 wt.% of a superparamagnetic metal oxide such as superparamagnetic iron oxide is produced by conjugating a superparamagnetic metal oxide such as superparamagnetic iron oxide surface-coated with silica with an inorganic porous wall material having an outer surface area of >=50 m<2> /g and composed of magnetic silica particles having a specific surface area of 10-800<2> /g and particle diameter of 0.5-15 μm. The surface pore diameter of the magnetic silica particle is 0.1-60 nm and the pore volume is 0.01-1.5 mL/g. The magnetic carrier can separate a nucleic acid from a bio-specimen, especially a whole blood specimen in high efficiency and is useful e.g. as a carrier for the automation of a nucleic acid isolation process.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a nucleic acid-binding magnetic carrier for isolating a nucleic acid from a sample suspected of containing the nucleic acid, a nucleic acid isolation reagent kit containing the carrier, and a nucleic acid isolation method.

[0002]

2. Description of the Related Art Recent advances in fields such as genetic engineering and molecular biology have made it possible to analyze infectious diseases and genetic diseases at the DNA or RNA level. In particular, PCR (Polymerase Chain Reaction, Sci.
ence 230: 1350-1354, 1985) and the NASBA method (Nucleic
Acid Sequence Based Amplification, Nature 350: 91-9
The invention of the nucleic acid amplification method represented by 2, 1991) has made it possible to detect a very small amount of nucleic acid, which has been extremely difficult to detect in the past, and has greatly facilitated gene analysis. .

However, in order to amplify and detect nucleic acids in a biological sample as necessary, it is necessary to selectively remove the nucleic acids from the sample. This is because ordinary biological samples contain a large amount of contaminants other than nucleic acids, for example, proteins, lipids, saccharides, and the like, and these have many possibilities of adversely affecting amplification and detection. Therefore, it is necessary to remove nucleic acids in the sample and to isolate the nucleic acid in advance.

[0004] This nucleic acid isolation method has been performed by various methods since ancient times. A representative example is the phenol method (Biochimica et Biophysica Acta, 72: 619-629, 196
3), alkali method (Nucleic Acid Research, 7: 1513-152
3, 1979). These are widely used on a laboratory scale, but require technical skills because they use dangerous organic solvents such as phenol and chloroform, which are difficult to dispose of, and sodium hydroxide, which is a dangerous substance. It is not easy to carry out with good reproducibility.

As a system using a nucleic acid-binding carrier for isolating a nucleic acid, a method using glass particles and sodium iodide (Proc. Natl. Acad. Sci. USA., 76-2: 615-619, 197)
9), a method using hydroxyapatite (Japanese Unexamined Patent Publication No.
No. 63093). These methods do not use harmful organic solvents, etc., but instead involve many centrifugation operations during the process, so it is difficult to process many samples at once, and it takes a long time to isolate nucleic acids Includes the problem.

Accordingly, the above-mentioned nucleic acid isolation method has drawbacks such as the use of dangerous reagents such as organic solvents and alkalis, the necessity of centrifugation, and the difficulty in treating a large number of samples. There is a point. It occurs in the mechanization of the extraction process. Automated mechanization of nucleic acid isolation is becoming indispensable in the future in order to process a large number of samples with good reproducibility and to further reduce human costs.

[0007] To achieve this automatic mechanization, a method using silica particles and chaotropic ions (J. Cli
nical Microbiology, 28-3: 495-503, 1990 and JP
2-289596). This involves mixing nucleic acid-binding silica particles and chaotropic ions capable of releasing nucleic acids in a sample with the sample, binding the nucleic acids to the silica particles, removing contaminants by washing, and then binding to the silica particles. The recovered nucleic acid is recovered. The method is DN
In addition to A, it is also suitable for extracting a more unstable RNA, and is very excellent in that a nucleic acid with high purity can be obtained. However, it is necessary to carry out centrifugation or filtration using a filter or the like for washing the particles to which the nucleic acid is bound, and there is a high possibility that the process becomes complicated during the mechanization.

As a method for easily mixing or washing silica particles, the use of magnetic silica particles is already known, and the present inventors have proposed the use of magnetic silica particles having a specific surface area to improve the non-magnetic properties. A magnetic particle carrier capable of specifically adsorbing many nucleic acids, and a method for effectively isolating nucleic acids from biological materials by a simple operation using this carrier (Japanese Patent Application Laid-Open No. H9-19292) have been proposed. . However, simply using magnetic particles having a specific specific surface area often causes a phenomenon in which the efficiency of nucleic acid recovery is reduced due to the influence of impurities in the sample on adsorption inhibition.

[0009]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above problems, and it is possible to efficiently isolate a nucleic acid from a biological sample, particularly a whole blood sample, Another object of the present invention is to provide a method capable of automatically mechanizing nucleic acid isolation.

[0010]

Means for Solving the Problems The present inventors have made intensive studies to solve these problems, and as a result, in order to efficiently recover nucleic acids from a biological sample, the magnetic silica particle carrier has at least 50 m ^2. / G of external surface area was found to be effective and arrived at the present invention.

That is, a nucleic acid binding magnetic carrier which is magnetic silica particles containing a superparamagnetic metal oxide, wherein the magnetic silica particles have an external surface area of at least 50 m ² / g. It is a carrier.

The present invention also provides a nucleic acid-binding magnetic carrier, a nucleic acid-adsorbing solution for adsorbing a nucleic acid to the nucleic acid-binding carrier, and a solution for eluting the nucleic acid from the nucleic acid-binding carrier-nucleic acid complex. It is a nucleic acid isolation reagent kit characterized by including:

Further, the present invention provides the following steps (a) to (c):
It is a nucleic acid isolation method characterized by including the following. (A) mixing the nucleic acid with the nucleic acid-binding carrier by mixing the nucleic acid-binding magnetic carrier, the sample containing the nucleic acid, and the nucleic acid adsorption solution for adsorbing the nucleic acid to the nucleic acid-binding carrier; Separating the nucleic acid-binding carrier to which is bound from the liquid, washing if necessary, and (c) elute the nucleic acid from the nucleic acid-binding carrier-nucleic acid complex

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Next, words used in the present invention are defined, and a method for measuring the words will be described. The surface area indicates the area on the entire surface of the carrier. Further, in the case of micro-sized particles, not the surface area per particle, but the unit weight (for example, 1 g)
It is often indicated by the surface area per unit, and this is called the specific surface area. The external surface area refers to a surface area located on the surface of the carrier.

[0015] The pores indicate fine cavities existing on the particle surface, and the pore volume indicates the volume of a space constituted by the pores. The surface pore diameter is the diameter of the fine pores present on the carrier, and the average diameter of the fine pore particles is often used. The particle diameter indicates a diameter when the shape of the dispersed particles is assumed to be spherical. These surface area pore diameters and pore volumes are measured by nitrogen gas adsorption measurement.

These measuring methods are based on JIS K1150
Standardized by the "Silica gel test method". Others are B
It is measured by an ET method, a nitrogen substitution (t-plot) method, or the like.

In general, the surface area of the magnetic silica particle carrier is divided into an internal surface area originating from the pores and an external surface area substantially appearing as a surface because the particles are porous. Then, in the present invention, they have found that the latter external surface area affects the efficiency of recovering nucleic acid in a sample.

That is, in the present invention, the magnetic silica particle carrier is at least 50 m ² / g, preferably 70 m ² / g.
It is necessary to have an external surface area of 100 m ² / g. By using particles having a larger external surface area, the amount of nucleic acid recovered can be increased. 50
If it is less than m ² / g, there is a disadvantage that contaminants in the sample bind to the particle surface and nucleic acids cannot be adsorbed.

In the present invention, the surface area of the particles is analyzed by the t method. In this method, the nitrogen adsorption isotherm of a sample to be analyzed is compared with that of a standard sample having similar surface characteristics. First, a standard t-curve is created from the adsorption isotherm of the non-porous material. Here, assuming that the thickness of the monolayer of nitrogen molecules is 0.354 nm, the thickness tnm of the adsorption amount of the standard sample using nitrogen gas is calculated. Then, the nitrogen adsorption amount of the analysis sample is plotted against the thickness of the reference sample adsorption amount. This t-plot takes the form as shown in FIG. 1 for the adsorbent having micropores shown in the present invention.

The magnetic silica particles of the present invention preferably have a specific surface area of 10 to 800 m ² / g and a particle size of 0.5 to 15 μm.
m, particularly preferably 1.0 to 10.0 μm. The magnetic silica particles of the present invention have a surface pore diameter of preferably 0.01 to 60 nm, particularly preferably 0.5 to 10 nm.
And the pore volume is preferably from 0.01 to
1.5 ml / g, particularly preferably 0.01 to 0.5 ml
/ G.

In a preferred embodiment, the magnetic silica particles of the present invention have the following structure. That is,
The surface of the following superparamagnetic metal oxide is coated with silica, and is further composited with an inorganic porous wall material composed of fine silica particles. The magnetic silica particles are almost perfectly spherical. The nucleic acid and the silica particles are bonded by a hydrogen bond generated between the hydroxyl group on the silica surface and the base of the nucleic acid.

The superparamagnetic metal oxide used in the present invention refers to a metal oxide which responds to the degree of change in the magnetic field but has no permanent magnetization and a small residual magnetization. Preferred superparamagnetic metal oxides include iron oxide. As the iron oxide, r-type iron sesquioxide (rFe ₂ O ₃ ) obtained by gradually oxidizing iron sesquioxide (Fe ₃ O ₄ ) and iron sesquioxide
Are used. This triiron tetroxide has a low remanence and a more favorable surface structure (substantially spherical), so that the cycle of magnetic separation and redispersion can be repeated. Magnetic silica particles containing ferric oxide,
It is stable in weakly acidic aqueous solutions and can be stored for more than 2 years.

The weight of the superparamagnetic metal oxide contained in the magnetic silica particles of the present invention depends on the strength of the magnetic pole.
６０60 wt%, more preferably 20-40.
wt%. Within this preferred range, the magnetic support can be rapidly separated using commercially available magnets.

In the present invention, silica includes SiO ₂ crystals and other forms of silicon oxide, a skeleton of a diatom plant composed of SiO _2, and amorphous silicon oxide.

The magnetic silica particles of the present invention most preferably have the following properties. (1) The carrier is magnetic silica particles containing superparamagnetic iron oxide. (2) The external surface area of the magnetic silica particles is at least 50 m
² / g. (3) The magnetic silica particles are composed of a superparamagnetic metal oxide whose surface is coated with silica and an inorganic porous wall material composed of fine silica particles. (4) The weight of the superparamagnetic iron oxide is 10 to 60 wt%. (5) The specific surface area of the magnetic silica particles is 10 to 800 m ² /
g. (6) The surface pore diameter of the magnetic silica particles is 0.1 to 60 n.
m. (7) The pore volume of the magnetic silica particles is 0.01 to 1.5 m.
1 / g. (8) The particle diameter of the magnetic silica particles is 0.5 to 15 μm.

The magnetic silica particles of the present invention are, for example,
It can be produced by the method described in JP-A-6-47273. For example, ferric oxide is added to an alcohol solution of tetraethoxysilane and dispersed and wetted by an ultrasonic disperser. A hydrolysis catalyst for tetraethoxysilane is added thereto, and silica is deposited on the surface of iron sesquioxide while being ultrasonically dispersed. Sodium silicate is added to the dispersion thus obtained, and an organic solvent and a surfactant (a solution of sorbitan monostearate in toluene) are added to emulsify to form a W / O emulsion. This emulsion is added to an aqueous solution of ammonium sulfate and thoroughly stirred. Thereafter, filtration separation, washing with water, alcohol precipitation and drying are performed to obtain desired spherical silica particles.

The reagent kit for nucleic acid isolation of the present invention comprises a nucleic acid-binding magnetic carrier, a nucleic acid-adsorbing solution for allowing nucleic acid to be adsorbed to the nucleic acid-binding carrier, and a nucleic acid-binding carrier-nucleic acid complex. Solution.

The nucleic acid adsorbing solution in the present invention is a solution having a function of destroying cells containing nucleic acids in a biological sample, exposing the nucleic acids, and binding the nucleic acids to magnetic silica particles. As such a solution, a substance that enhances the hydrophobicity of the silica particle surface, such as a chaotropic substance, is preferably selected. Further, a nucleic acid, such as guanidine (iso) thiocyanate and / or guanidine hydrochloride, is preferably used. A solution of a compound having a degrading enzyme inhibitory activity can be used.

Specific examples of the chaotropic substance include guanidine thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, sodium perchlorate, and urea. Of these, ribonuclease that degrades RNA It is preferable to use guanidine thiocyanate or guanidine hydrochloride, which has a large inhibitory effect on guanidine. The concentration used of these chaotropic substances differs depending on the chaotropic substance used, and is usually 1 to 8
M, preferably 4-7M, for example when using guanidine hydrochloride it is 4-7.5M. When guanidine thiocyanate is used, it is 3-5.5.
M range.

The nucleic acid-adsorbing solution containing the adsorbent of the present invention preferably contains a buffer. This may be contained in the solution for adsorption in advance, or may be added as a buffer after lysing the cells. The buffer is not particularly limited as long as it is generally used, but a buffer having a buffer capacity at around neutral, that is, at a pH of 5 to 9 is preferable. For example, tris-hydrochloride, sodium tetraborate-hydrochloric acid, potassium dihydrogen phosphate-sodium tetraborate buffer, and the like are used.
500 mM, pH is preferably in the range of 7-9.

Further, the nucleic acid adsorbing solution may contain a surfactant for the purpose of disrupting cell membranes or denaturing proteins contained in cells. The surfactant is not particularly limited as long as it is generally used for nucleic acid extraction from cells or the like. Specifically, nonionic surfactants such as triton-based surfactants and tween-based surfactants are used. And an anionic surfactant such as sodium N-lauroyl sarcosine. In the present invention, it is particularly preferable to use a nonionic surfactant in the range of 0.1 to 2%.

Further, the nucleic acid adsorbing solution preferably contains a reducing agent such as 2-mercaptoethanol or dithiothreitol for the purpose of denaturing and inactivating proteins contained in the sample, particularly ribonuclease.

The washing solution optionally used in the present invention is not particularly limited as long as it does not promote elution of the nucleic acid from the nucleic acid binding carrier and prevents binding of proteins, saccharides and lipids to the solid phase. Not limited. In particular,
4-7.5M guanidine hydrochloride solution or 40-80
It is preferred to wash with% ethanol. Also, if the adsorption solution used in the dissolution / adsorption step is used as a washing solution, it is effective for removing genomic DNA and proteins. At this time, it is preferable to subsequently wash with 40 to 80% ethanol.

The nucleic acid extract for eluting a nucleic acid from a nucleic acid-binding carrier to which the nucleic acid is bound in the present invention is not particularly limited as long as it promotes elution of the nucleic acid from the nucleic acid-binding carrier. Specifically, water or TE buffer (10
mM Tris-HCl buffer, 1 mM EDTA: pH8.
0) is preferred. When forming a nucleic acid-magnetic silica particle complex, when processing a biological sample containing a large amount of contaminants, for example, a sample such as blood, a substance derived from the sample adheres around the magnetic silica particles, They tend to prevent binding. In fact, the sample with more contaminants has lower nucleic acid recovery ability. In order to solve this problem, in the present invention, by using particles having a large external surface area, even if the contaminant derived from the sample adheres to the particles, it is unlikely to affect the binding of the nucleic acid, and therefore, high Recovery efficiency can be maintained.

The nucleic acid isolation reagent kit of the present invention comprises (1)
A nucleic acid-binding magnetic carrier, which is a magnetic silica particle containing a superparamagnetic metal oxide, wherein the magnetic silica particle has an external surface area of at least 50 m ² / g. And (3) a solution for eluting nucleic acids.
The composition ratio is variously selected depending on the purpose of use,
For example, (1) about 20 to 200 μl, (2)
About 100 to 10,000 μl and (3) about 20 to 20
There is 0 μl.

In the present invention, such magnetic silica particles are added to a solution for nucleic acid adsorption together with a nucleic acid-containing sample to form a magnetic silica particle-nucleic acid complex. The nucleic acid isolation method of the present invention specifically includes the following steps. (A) mixing a nucleic acid-binding carrier, a sample containing the nucleic acid, and a nucleic acid adsorbing solution for adsorbing the nucleic acid to the nucleic acid-binding carrier, and binding the nucleic acid to the nucleic acid-binding carrier (adsorption step); ) A step of separating the nucleic acid-binding carrier to which the nucleic acid is bound from the liquid and washing if necessary (separation step); and (c) a step of eluting the nucleic acid from the nucleic acid-binding carrier-nucleic acid complex (elution step)

Here, the sample containing nucleic acid includes whole blood,
It includes biological materials derived from animals such as serum, plasma, urine, saliva, body fluids, and other non-animal biological materials such as plants and microorganisms. It also includes cells and cultured cells isolated from these organisms. Furthermore, a partially purified nucleic acid is also included. Nucleic acid means DNA or RNA, and DNA is double-stranded DNA, single-stranded DNA, plasmid DN.
A, genomic DNA, cDNA, etc. Also, RNA
As well as endogenous RNAs derived from organisms producing these biological materials, in addition to RNAs derived from foreign parasites such as viruses, bacteria, and fungi.
-RNA, r-RNA and the like.

In the (a) adsorption step of the present invention, the nucleic acid-binding carrier, the nucleic acid-containing sample and the nucleic acid adsorbing solution are mixed, and the nucleic acid is adsorbed on the nucleic acid-binding carrier. The mixing method is
There are vortex stirring, inversion mixing, magnetic stirring and the like, and the mixing time is about 5 to 60 minutes. By mixing these substances, nucleic acids, proteins, saccharides and the like in the sample are physically adsorbed to the nucleic acid binding carrier.

(B) As a means for separating the liquid phase and the nucleic acid binding carrier in the separation step, a superparamagnetic metal oxide in the particles is used, so that a simple magnetic separation method using a magnet or the like is possible. . (B) In the separation step, washing is performed as necessary to elute unnecessary proteins, saccharides, lipids, and the like. Washing is performed once or twice or more.

The (c) elution step is a step of eluting the nucleic acid from the nucleic acid binding carrier to which the nucleic acid has been adsorbed in the above (b) step. The nucleic acid recovered at this time can be directly used for an enzymatic reaction using a restriction enzyme, a DNA polymerase, or the like without performing a desalting or concentration operation such as dialysis or ethanol precipitation. After amplification as required, the target nucleic acid can be detected using a nucleic acid probe reagent.

[0041]

Next, the present invention will be described by way of examples.
The effect is made clearer. However, these
The scope of the present invention is not limited by the examples.
No. Example 1 The magnetic silica particles have an average particle diameter in the range of 1 to 10 μm,
The content of ferric oxide particles is 30% per gram weight,
Specific surface area is 50-500m^Two / G (Suzuki Yushi
Was used (see Table 1).

First, the above magnetic silica particles suspended in sterilized water so as to have a concentration of 0.5 g / ml were prepared. Under these conditions, the external surface area is 10 to 70 m ² / g.
To some extent, several kinds of different magnetic silica particles were prepared. Table 1 shows the properties of the magnetic silica particles in each Lot.

As a biological sample, a methicillin-resistant Staphylococcus aureus (hereinafter referred to as MRSA) -positive whole blood sample was used. In addition, as a solution for nucleic acid extraction, 5 mol / l
Guanidine thiocyanate, 1.0% Triton X-10
0 and 20 mmol / l ethylenediaminetetraacetic acid (E
DTA) was used. As a washing solution, a Tris / HCl buffer containing 5 mol / l guanidine thiocyanate was used. A 70% aqueous ethanol solution and acetone were used to remove high-concentration salts contained therein, and sterile water was used as an eluent for recovering nucleic acids bound to the particle carrier.

As specific operations, the following (1) to (1)
Step 3) was performed. (1) 0.9 ml of the nucleic acid extraction solution was placed in a 1.5 ml Eppendorf tube.
ml was added and mixed well. (2) Next, magnetic silica particles suspended in sterilized water 0.1 m
1 was added. (3) Mix well and leave at room temperature for 10 minutes. (4) The above-mentioned tube was set on a magnetic pole stand dedicated to a 1.5 ml Eppendorf tube to move particles to the tube wall surface on the magnetic pole side. (5) The solution phase was suctioned and discharged using a filter tip or a disposable dropper. (6) The tube was taken out of the magnetic pole stand, and 1.0 ml of a Tris / hydrochloric acid buffer containing 5 mol / l guanidine thiocyanate was added as a washing solution. (7) After sufficient mixing, set on a dedicated magnetic pole stand,
The solution was drained as above. (8) The washing operation with the washing solution was repeated once. (9) The nucleic acid-adsorbed magnetic silica particles were washed with 1.0 ml of a 70% ethanol solution in the same manner as described above to remove high-concentration salts. (10) After washing again with 1.0 ml of a 70% ethanol solution, washing was similarly performed with 1.0 ml of acetone. (11) The above tube was placed on a heat block at about 56 ° C., and left for about 10 minutes to completely evaporate and remove acetone in the tube and in the magnetic silica particles. (12) 0.1 ml of sterilized water was added, the tube was placed on a heat block at about 56 ° C., and left for 10 minutes. (13) The solution phase was set on a magnetic pole stand, the solution phase was sucked with a filter tip, and collected in another new tube. The recovery amount is usually about 60 to 70 μl.

Next, the recovered nucleic acid solution was added to NASBA.
Amplification was carried out by the method (Nature 350: 91-92, 1991, Japanese Patent No. 2648802 and Japanese Patent No. 2650159). That is, amplification uses two types of primers (SEQ ID NOs: 1 and 2) having an optimal sequence from the sequence of the mecA gene,
T7 RNA polymerase, reverse transcriptase and RNase
H (ribonuclease that hydrolyzes RNA of RNA-DNA hybrids without hydrolyzing single- or double-stranded RNA or DNA) at 41 ° C.
The amplification reaction was performed for 0 minutes.

The nucleic acid was detected by dot blot hybridization using a nylon membrane as a solid phase. The nucleic acid solution obtained by the above amplification reaction was diluted to 10-fold concentration with sterile water, and 5 × SS
C (0.075 mol / l Na-Citrate, pH 7.0, 0.75 mol / l NaCl
1.0 μl was blotted on a nylon membrane washed with (mixture). After drying, the membrane was placed in a hybrid bag (manufactured by BRL), and 1.0 ml of a hybridization buffer containing a detection probe (SEQ ID NO: 3) labeled with alkaline phosphatase was added thereto.
Hybridization was performed for minutes. Thereafter, the membrane was removed from the hybrid bag, and the washing solution 1 (5 × SSC,
(1.0% sodium lauryl sulfate) and transferred to a vat and washed at 50 ° C. for 10 minutes. Next, cleaning solution 2 (5 × S
(SC, 0.5% Triton X-100) and washed at room temperature for 10 minutes while shaking. The washed membrane is placed in a new hybrid bag, sealed with a substrate solution of alkaline phosphatase containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate, and left standing at 37 ° C. for 60 minutes. A color development reaction was performed. After color development, the membrane is washed with distilled water and dried,
Color development is color difference meter CR-221 (Minolta Camera)
Was measured by The ΔE mode was used for the measurement.

As is clear from Table 1 below, 5
When magnetic silica particles having an external surface area of 0 m ² / g or more (LotA in Table 1) are used for nucleic acid extraction and isolation, magnetic particles having a small external surface area (LotD, G, H in Table 1) should be used. The detection value was clearly higher than when used. This indicates that the nucleic acid extraction efficiency has increased.

[0048]

[Table 1]

[0049]

The present invention, which uses magnetic silica particles having an external surface area of at least 50 m ² / g as a carrier for extracting and isolating nucleic acids, is excellent in recovering nucleic acids. Especially,
In the processing of a sample containing a large amount of contaminants, for example, a blood sample, it is excellent in the recovery amount.

[0050]

[Sequence List] SEQ ID NO: 1 Sequence length: 21 Sequence form: Nucleic acid Topology: Single-stranded Sequence type: Synthetic DNA Sequence characteristics Location: 1..21 Method for determining characteristics: S Other characteristics : Having a sequence homologous to the nucleotide sequence of positions 1332 to 1352 of the mecA gene of S. aureus. Sequence CGTTACAAGA TATGAAGTGG T 21

SEQ ID NO: 2 Sequence length: 47 Sequence form: Nucleic acid Topology: Single-stranded Sequence type: Synthetic DNA Sequence characteristics Location: 1..47 Method for determining characteristics: S Other characteristics: It has a sequence complementary to the nucleotide sequence at positions 1552 to 1571 of the mecA gene of S. aureus. It also has a promoter sequence for T7 RNA polymerase. Sequence AATTCTAATA CGACTCACTA TAGGGAGCCT TGTCCGTAAC CTGAATC 47

SEQ ID NO: 3 Sequence length: 25 Sequence form: Nucleic acid Topology: Single-stranded Sequence type: Synthetic DNA Sequence characteristics Location: 1..25 Method for determining characteristics: S Other characteristics: It has a sequence homologous to the nucleotide sequence at positions 1410 to 1434 of the mecA gene of S. aureus. Sequence TAGAGTAGCA CTCGAATTAG GCAGT 25

[Brief description of the drawings]

FIG. 1 is a diagram showing t-plot.

Claims (9)

[Claims] 1. A nucleic acid binding magnetic carrier comprising magnetic silica particles containing a superparamagnetic metal oxide, wherein the magnetic silica particles have an external surface area of at least 50 m ² / g. Carrier. 2. The magnetic silica particle carrier has a particle diameter of 70 to 100 m.
The nucleic acid binding magnetic carrier according to claim 1, which has an external surface area of ² / g. 3. The nucleic acid according to claim 1, wherein the magnetic silica particles are a composite of a superparamagnetic metal oxide whose surface is coated with silica and an inorganic porous wall material composed of fine silica particles. Binding carrier. 4. The method according to claim 1, wherein the magnetic silica particles have a particle diameter of 10 to 800 m ² /
The nucleic acid binding magnetic carrier according to claim 1, which has a specific surface area of g. 5. The nucleic acid-binding magnetic carrier according to claim 1, wherein the magnetic silica particles have a particle size of 0.5 to 15 μm. 6. The magnetic silica particles have a particle size of 1.0 to 10.0 μm.
The nucleic acid-binding magnetic carrier according to claim 1, which has a particle diameter of m. 7. A nucleic acid-binding magnetic carrier having the following properties (1) to (7). (1) The carrier is magnetic silica particles containing superparamagnetic iron oxide. (2) The external surface area of the magnetic silica particles is at least 50 m
² / g. (3) The magnetic silica particles are composed of a superparamagnetic metal oxide whose surface is coated with silica and an inorganic porous wall material composed of fine silica particles. (4) The weight of the superparamagnetic iron oxide is 10 to 60 wt%. (5) The specific surface area of the magnetic silica particles is 10 to 800 m ² /
g. (6) The surface pore diameter of the magnetic silica particles is 0.1 to 60 n.
m. (7) The pore volume of the magnetic silica particles is 0.01 to 1.5 m.
1 / g. (8) The particle diameter of the magnetic silica particles is 0.5 to 15 μm. 8. A nucleic acid-binding magnetic carrier according to claim 1, a nucleic acid-adsorbing solution for adsorbing a nucleic acid to the nucleic acid-binding carrier, and a nucleic acid-binding carrier-nucleic acid complex. A reagent kit for nucleic acid isolation, comprising a solution for eluting a nucleic acid from a nucleic acid. 9. A method for isolating a nucleic acid, comprising the following steps (a) to (c). (A) mixing the nucleic acid-binding magnetic carrier according to any one of claims 1 to 7, a nucleic acid-containing sample, and a nucleic acid-adsorbing solution for allowing the nucleic acid to be adsorbed to the nucleic acid-binding carrier; (B) separating the nucleic acid-binding carrier to which the nucleic acid has been bound from the liquid, washing if necessary, and (c) elute the nucleic acid from the nucleic acid-binding carrier-nucleic acid complex