JP3395227B2 - Nucleic acid hybridization assay - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a nucleic acid hybridization assay method. More specifically, the present invention provides a nucleic acid hybridization measurement method with high sensitivity and less non-specific reaction by allowing a glycoside derivative and / or a glycoside derivative-containing (co) polymer to exist in a nucleic acid hybridization reaction system or its washing system. Regarding how to achieve.

[0002]

2. Description of the Related Art In recent years, a nucleic acid hybridization assay has been widely used to detect or quantify a target nucleic acid having a specific sequence from a sample derived from various biological components. This method utilizes the property that single-stranded denatured nucleic acids try to bind to each other via hydrogen bonds with single-stranded nucleic acids having complementary sequences under appropriate conditions. As a method of measuring by such a nucleic acid hybridization reaction, a method of labeling a nucleic acid with a radioisotope, a method of using an enzyme-labeled nucleic acid and the like are known. In addition, a nucleic acid hybridization method in which a nucleic acid is carried on a carrier such as a membrane, latex particles, or a microtiter plate and a target nucleic acid in a sample is detected has been widely used. In recent years, Ranki etc.
et.al.), Gene. 21: P77-85 (1983) and JP-A-6
2-205800 discloses a DNA probe sandwich assay (hereinafter referred to as sandwich hybridization) method. In this method, one probe is immobilized on a membrane, hybridized with a target nucleic acid having a specific sequence, and a second probe for detection that can hybridize with another sequence portion of the target nucleic acid is added. It consists of reacting. A more detailed description is disclosed in JP-A-60-93355.

In these measuring methods using nucleic acid hybridization reaction, various additives are used for the purpose of promoting the hybridization reaction or effectively measuring a trace amount of components. For example, polyethylene glycol (PEG) is used as a reaction system (Japanese Patent Laid-Open No.
-47256, JP-A-59-54968,
JP-A-63-45066), hydrophilic polymers such as dextran, bovine serum albumin (BSA), or sodium lauryl sulfate (SDS) (T. Maniatis et.a.
l., Molecular Cloning, 1989.P9.49, P11.50 ~ 11.57).
A method of adding a surfactant such as polyvinylpyrrolidone (PVP) or the like has been adopted.

[0004]

However, the sample used as the specimen has various compositions and properties, and therefore many hybridization reactions involve many nonspecific reactions which should be called side reactions unrelated to the reaction of nucleic acids. It is known. The use of such additives often enhances the reaction between the target nucleic acid and the nucleic acid thereto, and at the same time, enhances the nonspecific reaction. In addition, even in the washing step, nonspecific binders cannot be sufficiently removed by the above additives, and as a result, the background value becomes high, and it should be detected that the measured value does not exceed the background value. I couldn't. That is, the detection sensitivity was low, and a large amount of sample was required to obtain a signal above a certain level. Therefore, this method cannot be said to be a sufficient method for promoting only the nucleic acid hybridization reaction and effectively measuring a trace amount component.
Also, when a surfactant such as sodium lauryl sulfate (SDS) is used, the solubility is low at low temperature,
When a solution having a concentration capable of obtaining a sufficient action is prepared, it precipitates at room temperature, so it is necessary to dissolve it by heating before use, which is a problem in operation. The present invention solves the above-mentioned conventional problems, and an object of the present invention is to provide a highly sensitive nucleic acid hybridization method in which a non-specific reaction is suppressed.

[0005]

As a result of intensive studies to solve these problems, the present inventors have found that in order to suppress non-specific reactions, glycosides are added to the reaction system of nucleic acid hybridization or its washing system. It has been found to be effective to have a water-soluble polymer and / or water-soluble copolymer containing a derivative and / or a glycoside derivative as a monomer unit. That is, the present invention is represented by the following general formula (I) in the reaction system or the washing system of the reaction in a nucleic acid hybridization reaction in which a nucleic acid that complementarily binds to a target nucleic acid that is a substance to be measured is reacted. A nucleic acid hybridization method characterized by the presence of at least one glycoside derivative.

General formula (I):

[0007]

[Chemical 5]

(In the formula, G represents a residue in which the OH group bonded to the carbon atom at the 1-position of the reducing terminal monosaccharide is removed from the saccharide. X represents an oxygen atom or a sulfur atom. P represents 1-18. Represents an organic group containing a carbon atom of.)

The present invention also relates to a nucleic acid hybridization reaction in which a nucleic acid that complementarily binds to a target nucleic acid, which is a substance to be measured, is reacted with the reaction system or the washing system of the reaction by the following general formula (IV). It is a nucleic acid hybridization method characterized in that a water-soluble polymer and / or a water-soluble copolymer containing at least one glycoside derivative as a monomer unit is present.

General formula (IV):

[0011]

[Chemical 6]

(In the formula, G represents a residue in which the OH group bonded to the carbon atom at the 1-position of the reducing terminal monosaccharide is removed from the saccharide. X represents an oxygen atom or a sulfur atom. R represents a hydrogen atom or methyl. Group or ethyl group, m represents an integer of 1 to 3, n
Represents an integer of 1 to 4. )

Examples of the glycoside derivative (I) used in the present invention include compounds represented by the following general formula (II) or compounds represented by the following general formula (III).

General formula (II):

[0015]

[Chemical 7]

(In the formula, G represents a residue in which OH bonded to the carbon atom at the 1-position of the reducing terminal monosaccharide is removed from the saccharide, X represents an oxygen atom or a sulfur atom, and Q represents a hydrogen atom or a hydroxy group. M is an integer of 1 to 3, n is 1 to 4
Indicates an integer. )

General formula (III):

[0018]

[Chemical 8]

(In the formula, G represents a residue in which an OH group bonded to the carbon atom at the 1-position of the reducing terminal monosaccharide is removed from the saccharide. X represents an oxygen atom or a sulfur atom. R represents a hydrogen atom or methyl. Group or ethyl group, m represents an integer of 1 to 3, n
Represents an integer of 1 to 4. )

Specific examples of the compound represented by the general formula (II) include the following compounds. (In the formula, G represents a residue in which the OH group bonded to the carbon atom at the 1-position of the reducing terminal monosaccharide is removed from the saccharide. Further, the following compounds are α-, β- or a mixture thereof. glycoside (G-O-CH ₃₎ ethyl glycoside _{(G-O-C 2 H} 5) propyl glycoside _{(G-O-C 3 H} 7) butyl glycoside _{(G-O-C 3 H} 6 -CH 3) hexyl glycoside _{(G-O-C 5 H} 10 -CH 3) octylglucoside _{(G-O-C 7 H} 14 -CH 3) nonyl glycoside _{(G-O-C 8 H} 16 -CH 3) dodecyl glycoside (G-O- C ₁₁ H ₂₂ -CH ₃₎ hydroxy methyl glycoside _{(G-O-CH 2 OH} ) 2- hydroxyethyl glycoside _{(G-O-C 2 H} 4 OH) 3- hydroxypropyl glycoside _{(G-O-C 3 H} 6 OH) 4-hydroxybub Chirugurikoshido _{(G-O-C 4 H} 8 OH) 6- hydroxyhexyl glycoside _{(G-O-C 6 H} 12 OH) 8- hydroxy octylglucoside _{(G-O-C 8 H} 16 OH) 9- hydroxy nonyl glucoside _{(G-O-C 9 H} 18 OH) 12- hydroxy dodecyl glycoside _{(G-O-C 12 H} 24 OH) methylthio glycoside (G-S-CH ₃₎ ethylthiomethyl glycoside _{(G-S-C 2 H} 5) propyl thio glycoside _{(G-S-C 3 H} 7) butyl thioglycoside _{(G-S-C 3 H} 6 -CH 3) hexyl thioglycoside _{(G-S-C 5 H} 10 -CH 3) octyl thioglycoside (G _{-S-C 7 H 14 -CH 3} ) Noni thio glycoside _{(G-S-C 8 H} 16 -CH 3) dodecyl thioglycoside _{(G-S-C 11 H} 22 -CH 3) hydroxymethyl thio Glycoside _{(G-S-CH 2 OH} ) 2- hydroxyethyl thio glycoside _{(G-S-C 2 H} 4 OH) 3- hydroxypropyl thioglycoside _{(G-S-C 3 H} 6 OH) 4- hydroxybutyl thioglycoside _{(G-S-C 4 H} 8 OH) 6- hydroxyhexyl thioglycoside _{(G-S-C 6 H} 12 OH) 8- hydroxyoctyl thioglycoside _{(G-S-C 8 H} 16 OH) 9- hydroxy Noni Lucio Glycoside (GSC ₉ H ₁₈ OH) 12-Hydroxydodecylthioglycoside (GSC ₁₂ H ₂₄ OH)

Examples of the water-soluble polymer and / or the water-soluble copolymer (IV) containing the glycoside derivative as a monomer unit include the following substances. These polymers have a molecular weight of approximately 10 ² to 10 ⁵ .

Substance Name Polyglycosyloxyethyl methacrylate Structural formula

[0023]

[Chemical 9]

(In the formula, G represents a residue in which the OH group bonded to the carbon atom at the 1-position of the reducing terminal monosaccharide is removed from the saccharide, and n represents an integer of 1 to 1000.)

Substance Name Polyglycosyloxyethyl acrylate Structural formula

[0026]

[Chemical 10]

(In the formula, G represents a residue in which the OH group bonded to the carbon atom at the 1-position of the reducing terminal monosaccharide is removed from the saccharide, and n represents an integer of 1 to 1000.)

Substance Name Polyglycosyloxyethyl α-ethyl acrylate Structural formula

[0029]

[Chemical 11]

(In the formula, G represents a residue in which the OH group bonded to the carbon atom at the 1-position of the reducing terminal monosaccharide is removed from the saccharide, and n represents an integer of 1 to 1000.)

Substance Name Polyglycosyloxyethoxyethyl methacrylate Structural formula

[0032]

[Chemical 12]

(In the formula, G represents a residue in which the OH group bonded to the carbon atom at the 1-position of the reducing terminal monosaccharide is removed from the saccharide, and n represents an integer of 1 to 1000.)

Substance Name Polyglycosylthioethylmethacrylate Structural formula

[0035]

[Chemical 13]

(In the formula, G represents a residue in which the OH group bonded to the carbon atom at the 1-position of the reducing terminal monosaccharide is removed from the saccharide, and n represents an integer of 1 to 1000.)

In the above glycoside derivative, G is a residue in which the --OH group bonded to the carbon atom at the 1-position of the reducing terminal monosaccharide of a saccharide having no protective group is removed. Specifically, it means a sugar residue having about 1 to 10 sugar units, preferably 1 to 3 monosaccharide or oligosaccharide residues. Specific examples of monosaccharides include hexasaccharides such as glucose, mannose, galactose, glucosamine, mannosamine and galactosamine, and pentasaccharides such as arabinose, xylose and ribose. Specific examples of oligosaccharides include disaccharides such as maltose and lactose, maltotriose, isomaltotriose and maltotetraose.

Specific compounds used in the present invention include octyl glucoside (trade name: Scratch OG) β-octyl thioglucoside (trade name: Scratch OS)
G) Polyglucosyloxyethyl methacrylate (trade name:
Scraf poly GEMA) β-isopropyl thiogalactoside (trade name: Scraf IPTG) β-octyl galactoside β-octyl maltoside β-octyl mannoside β-decyl glucoside β-decyl galactoside β-decyl maltoside and the like.

The method for producing the glycoside derivative used in the present invention includes the following methods. Synthetic Method of Glycoside Derivative (III) (Part 1) Glycoside derivative (III) is prepared by the following general formula (V) in the presence of a heteropolyacid and a polymerization inhibitor:

[0040]

[Chemical 14]

(In the formula, G represents a residue in which the OH group bonded to the carbon atom at the 1-position of the reducing terminal monosaccharide is removed from the saccharide. X represents an oxygen atom or a sulfur atom. R ₁ represents the number of carbon atoms. 1 to
4 is a lower alkyl group. ) An alkyl glycoside represented by

The following general formula (IIIa):

[0043]

[Chemical 15]

(Wherein R represents a hydrogen atom, a methyl group or an ethyl group, m represents an integer of 1 to 3, and n represents an integer of 1 to 4). It can be produced by reacting. The reaction can be carried out in a solvent or without a solvent. The lower alkyl group as R ₁ in the above general formula (V) is a methyl group,
Ethyl group, propyl group, isopropyl group, butyl group, te
It means a linear or branched alkyl group having about 1 to 4 carbon atoms such as rt-butyl group and isobutyl group.

The starting compound, alkyl glycoside (V), is not particularly limited, and those produced by a known method or commercially available ones may be used. For example, an alkyl glycoside whose alkyl moiety is a linear or branched alkyl group having about 1 to 4 carbon atoms can be used, such as methyl glucoside, methyl β-D-galactoside, methyl-D-maltoside, and butylmannoside. The alkyl glycosides (V) may be used alone or in combination of two or more.

The other raw material, acrylic ester (IIIa), is not particularly limited, and known ones can be used, for example, 2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate, methacrylic acid-2. -Hydroxypropyl, 2-hydroxyethyl ethyl acrylate and the like can be mentioned, but not limited thereto. The type and amount of the heteropolyacid used are not particularly limited. The polymerization inhibitor is not particularly limited, and known ones can be used.

Synthetic Method of Glycoside Derivative (III) (Part 2) In addition, the glycoside derivative (III) is a sugar and an acryl which do not have a protecting group while supplying oxygen to the reaction system in the presence of an acid catalyst and a polymerization inhibitor. It can also be produced by reacting with an acid ester. According to this method, without using an alkyl glycoside which is relatively difficult to synthesize or obtain,
The target glycoside derivative (III) can be obtained in high yield by using an available sugar having no protecting group. As the saccharides, any of the monosaccharides and oligosaccharides mentioned above can be used without particular limitation as long as they have no protective group. As the acrylic ester, the above general formula (IIIa)
The one represented by can be used. The acid catalyst is not particularly limited and known ones can be used. As the polymerization inhibitor, known compounds can be used as in the case of the above-mentioned synthesis method (1). The method of supplying oxygen to the reaction system is not particularly limited, and for example, a gas containing oxygen such as air or oxygen may be blown into the reaction mixture.

Separation and Purification Method The glycoside derivative (III) obtained by the above reaction can be purified by a conventional purification means such as silica gel chromatography or extraction separation.

Method for Preparing Glycoside Derivative-Containing (Co) Polymer Further, a homopolymer comprising a repeating unit (IV) using the derivative represented by the general formula (III) as a monomer,
Polymerization of a copolymer composed of two or more different repeating units (IV) and a copolymer composed of the repeating unit (IV) and a repeating unit copolymerizable therewith can be carried out according to a usual method. Specific examples thereof include a bulk polymerization method, a solution (or uniform) polymerization method, a suspension polymerization method, an emulsion polymerization method, and a radiation (γ ray, electron beam) polymerization method. For example, in solution polymerization, the use ratio of the solvent, the polymerization component, the polymerization initiator and the solvent is not particularly limited. Also, two or more different glycoside derivatives (III)
In the case of copolymerizing, and also in the case of copolymerizing the glycoside derivative (III) and a compound copolymerizable therewith, the use ratio thereof is not particularly limited, and is appropriately selected depending on the use of the copolymer to be obtained and the like. do it. The compound copolymerizable with the glycoside derivative (III) is preferably a hydrophilic compound having an α, β unsaturated double bond, and examples thereof include acrylamide, acrylic acid, methacrylic acid and N-vinylpyrrolidone. . After completion of the polymerization, the desired polymer can be separated and collected from the polymerization reaction product and purified by a conventional means. The resulting polymer may then be further purified, for example by the reprecipitation method. The polymer thus obtained has a molecular weight of about 10 ² to 10 ⁵ .

Application to nucleic acid hybridization measurement method Background reduction effect The above glycoside derivative and / or the polymer and / or copolymer containing the glycoside derivative as a monomer are added to the nucleic acid hybridization measurement system at an appropriate concentration. When it does, it shows the effect of specifically reducing the background in the hybridization reaction. The largest cause of background in hybridization is nonspecific binding of substances other than the target to a carrier or the like. By preventing this reaction, an effect of reducing the background can be expected, and as a result, an increase in sensitivity can also be achieved.

The use of the substances according to the invention effectively prevents non-specific binding to the carrier or the like. Further, it also has an action of releasing even those non-specifically bound to the carrier or a similar substance. Specifically, the background measured value is reduced to about 1/3 to 1/10 as compared with the case of using a conventional surfactant or the like. At this time, if the measured value of the signal is at the same level, the signal / noise ratio is similarly increased by 3 to 10 times due to the reduction of the background. Therefore,
By using the substance according to the present invention, it is possible to specifically reduce the background in the hybridization reaction and, as a result, increase the sensitivity.

Applicable nucleic acid hybridization assay methods include commonly used hybridization methods, specifically dot blot hybridization, slot blot hybridization, sandwich hybridization, Southern blot hybridization, and Northern blot hybridization. It can be used for methods such as blot hybridization. As the carrier to be used, a membrane carrier such as nitrocellulose or nylon, latex particles, a microtiter plate and the like can be used. And the substance of the invention is
They can be used alone or in a mixture in the same manner as the commonly used additives such as bovine serum albumin or sodium lauryl sulfate. The final concentration in the solution is 10 when added to the reaction solution or washing solution.
% Or less, preferably 0.1 to 5.0%, more preferably 1.0
It is desirable to add it at a concentration of ˜2.0%, but not limited to this.

As the system for measuring the substance to be measured according to the present invention, a system in which both the substance to be measured and the reactant are soluble is used, that is, a so-called solution system, and the reactant is substantially insoluble in the reaction medium. It is applied to any so-called carrier system in which the carrier is sensitized (supported). It can also be used in a system in which a substance to be measured is directly bound to a carrier.
Here, as a method for supporting the reaction product, a method using a biotin-avidin system, a method using an antigen-antibody, a method using a hapten-antibody, a method of physically adsorbing, and sharing using a functional group of the reaction product A method of combining them can be used. As the labeling substance for nucleic acid, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or the like can be used. As a labeling method, labeling using a radioisotope (Richardson, Proceedings of the National Academy
of Sciences of USA Vol.54, P158.1968, Rigby et.
al., Journal of Molecular Biology, Vol.113, P237-25
1.1977.), A method for labeling with an enzyme (JP-A-60-9335).
No. 5) and the like. As the carrier to be used, one having a film-like structure, one having a particle structure, one formed of a polymer or the like can be used. As a material, nitrocellulose, nylon or the like can be used as the membranous carrier, and latex, polyethylene, polystyrene, polypropylene, polycarbonate or the like can be used as the particle or polymer molded product.

As a method for preparing the nucleic acid probe to be used,
A synthetic method such as the phosphoamidite method, a method in which bacteria are retained in the form of a plasmid, which is collected after growth and treated with various modifying enzymes such as restriction enzymes can be used. In addition, a method of directly introducing a label into the substance to be measured when preparing the substance to be measured can also be used. Specifically, a PCR method (Saiki et.al., S, which is one of gene amplification methods)
Cience. Vol. 230, P1350-1354, 1985.), a method of labeling by incorporating a labeled nucleotide such as biotin and incorporating it.

Measurement Item According to the present invention, any biological substance that can be generally measured by utilizing a nucleic acid hybridization reaction can be measured. The substance to be measured includes feces, urine, blood, sputum, and the like, and examples thereof include nucleic acid extracts and amplification products from various bacterial cells, viruses and the like. The nucleic acid extraction method is the phenol method (Saito et.al., Biochimica et Bi
ophysica Acta, Vol.72, P619-629, 1963.), and as the amplification method, PCR method (Saiki et.al., Science. Vol.230, P.
1350-1354, 1985.). Specific examples of the bacterial cells and viruses include, but are not limited to, Vibrio parahaemolyticus, cholera, dysentery, hepatitis virus, herpes virus, papilloma virus and the like.

[0056]

INDUSTRIAL APPLICABILITY In the present invention, a water-soluble polymer and / or a water-soluble copolymer containing the above-described specific glycoside derivative and / or glycoside derivative as a monomer unit is used in a nucleic acid hybridization measurement system (reaction system or washing system). By adding it, the non-specific reaction in the hybridization reaction can be reduced, and it becomes about 1/3 to 1/10 in terms of measured value. Moreover, when reducing the non-specific reaction, it does not affect the target specific reaction. Therefore, the detection sensitivity of the nucleic acid hybridization assay can be increased about 3 to 10 times. Further, this substance does not precipitate at a low temperature unlike the substances used conventionally at a concentration that brings about its effect, and there is no problem in its use. In addition, this substance has an application example of background reduction in the immunoassay method (Japanese Patent Laid-Open No. 4-122858), but there is no actual case where it is used for nucleic acid hybridization. It can be inferred that the effect of a surfactant such as this substance is very large in an irreversible reaction which is usually observed between an antigen and an antibody. However, in a reversible reaction such as nucleic acid hybridization, and even in a relatively low energy bonding reaction such as hydrogen bonding,
There is no predictability that this substance can be used as well.

[0057]

EXAMPLES The effects of the present invention will be further clarified by giving Examples and Comparative Examples of the present invention below, but the scope of the present invention is not limited by these Examples. In the examples, “n × SSC” is 0.3M Na-Citr
ate (pH 7.0), (20 / n) -fold diluted solution of 3.0 M NaCl mixture.

Reference Example 1 Synthesis of capture probe and labeled probe for detection of Vibrio parahaemolyticus The capture probe and labeled probe were DNA synthesizer 392.
Type (Applied Biosystems) was used to synthesize by the phosphoramidite method. The base sequence of the capture probe is 5'-CGGTCATTCTGCTGTGTTCGTAAAAT-3 '(SEQ ID NO: 1 in the Sequence Listing, below). The base sequence of the labeled probe is 5'-CCCCGGT
It is TCTGAXGAGATATTGTT-3 '(SEQ ID NO: 2 in the Sequence Listing below). In the sequence, X represents uridine having a linker arm at the 5-position. This labeled probe is disclosed in Japanese Patent Laid-Open No. 4-20299.

Reference Example 2 Labeling of Labeled Probe with Enzyme / Alkaline Phosphatase The binding between the labeled probe synthesized in Reference Example 1 and alkaline phosphatase via its linker arm was analyzed by literature (Nucleic Acids Research, 14, 6155, 1986). ). Linker oligonucleotide (labeled probe)
1.0 A260 was dissolved in 60 μl of 0.2 M NaHCO _3, and 1.25 mg of disuccinimidyl suberate (DSS) was added thereto and reacted at room temperature for 2 minutes. Add 1 mM CH ₃ COONa (pH 5.0) to the reaction mixture.
Equilibrated with. Then, gel filtration was performed using a Sephadex G-25 (Pharmacia) column (1 cm φ × 30 cm) to remove excess DS.
S was removed. A linker oligonucleotide in which the terminal amino group is activated is further added with a molar ratio of twice the alkaline phosphatase (Boehringer Mannheim) (10
Dissolved in 0 mM NaHCO ₃ and 3M NaCl. ) For 16 hours at room temperature to obtain an alkaline phosphatase-labeled nucleic acid probe. The obtained labeled probe was purified using negative ion exchange high performance liquid chromatography MONO-QFPLC (Pharmacia). Fractions containing the labeled probe were collected and concentrated by an ultrafiltration method using Centricon 30K (Amicon).

Reference Example 3 Preparation of Vibrio parahaemolyticus Gene A strain of Vibrio parahaemolyticus was treated with Brain Heart Infusion Agar containing 3% NaCl.
Were inoculated and cultured overnight at 37 ° C. Each grown colony is scraped into a 1.5 ml Eppendorf tube and 300 μl of dilution buffer (0.1M NaH ₂ PO ₄ , pH 7.0) is added.
Suspended in. Further, proteinase K (Nacalai Tesque, Inc.) 0.6 mg and lysate (8 M urea, 0.25% sodium dodecyl sulfate, 0.25% sodium lauryl sarcosine, 50 mM EDTA, pH 7.6) (600 μl) were added and stirred, and the mixture was stirred at 60
Incubated at 30 ° C for 30 minutes. The resulting solution was extracted twice with phenol and once with chloroform, followed by ethanol precipitation to obtain a nucleic acid.

Example 1 (1) Method for binding capture probe to solid phase As a solid phase, a polystyrene microtiter plate (Microlite 2, Dynatech) was used. The capture probe obtained in Reference Example 1 was diluted with 50 mM H ₃ BO ₃ buffer
Diluted to 0 pico mole / ml. 100 μl of the solution was dispensed into wells of a microtiter plate and incubated overnight at room temperature. (2) Blocking method of capture probe binding plate The capture probe solution was removed from the plate obtained by this method using an aspirator, and the block buffer was added to each well.
Dispense 150 μl each and block at room temperature for 2 hours. (3) Preparation of hybridization buffer containing additives Hybridization buffer (5 x SSC, 0.5%
Polyvinylpyrrolidone K-30) in which the glucoside derivative of the structural formula shown in Table 1 (trade name: Scraf, manufactured by Nippon Seika)
Was added at a concentration of 1.0% (W / V) to prepare a solution.

[0062]

[Table 1]

As a comparative example, a hybridization buffer containing sodium lauryl sulfate (special reagent for studying poorly soluble protein, Nacalai Tesque, Inc.) as an additive instead of various scrafs was prepared.

(4) Detection of Vibrio parahaemolyticus Gene by Sandwich Hybridization Method Including Various Additives in Hybridization Buffer Using the reagents and samples prepared by the methods of Reference Examples 2 and 3,
The detection of the Vibrio parahaemolyticus gene as a positive sample was performed by the method described below. Add an equal volume of 0.6N NaOH to the sample at room temperature.
It was denatured for 15 minutes. As a control (negative sample), a human placenta-derived DNA (Sigma) denatured by the same method was used. After binding the capture probe, remove the blocking solution from the blocked plate and add 2 μl of denatured sample.
Was added. Next, hybridization buffer (5 x S
SC, 0.5% Polyvinylpyrrolidone K-30, 1.0%
100 μl of various additives) were added, and hybridization was performed at 50 ° C. for 60 minutes. Remove the solution from the wells, and wash with washing solution (2 x SSC, 1.0% sodium lauryl sulfate).
μl was added and the mixture was washed at 50 ° C. for 5 minutes. Subsequently, the washing solution was removed, the well was rinsed with 200 μl of 1 × SSC, 100 μl of alkaline phosphatase-labeled probe solution was added, and
Hybridization was performed at 60 ° C. for 60 minutes. Remove the probe solution from the wells, add 200 μl of wash solution, and add 50 ° C.
Rinse for 10 minutes, then wash solution (1 x SSC, 0.5% T
ritonX-100) was added in an amount of 200 μl and washed at room temperature for 10 minutes. Finally, after adding 200 μl of 1xSSC and rinsing the well, Lumiph, a substrate for alkaline phosphatase, was added.
Add 100 μl of os480 (Wako Pure Chemical Industries, Ltd.), 15 at 37 ℃ in the dark.
A luminescence reaction was performed for a minute. Micro light 1000
(Dynatech). As is clear from Table 2 below, when the reagent of the present example is used, the value (background) measured for the negative sample is decreased and the value measured for the positive sample is increased as compared with the comparative example. Excellent results were obtained in terms of sensitivity and specificity, such as the appearance of

[0065]

[Table 2]

Example 2 (1) Method for binding capture probe to solid phase Magnetic beads (Dynal beads; Dynabead M-2) as a solid phase
80 Tosyl Activated) was used. Activated beads 1 mM HCl
The suspension was washed once with sterile distilled water. The capture probe obtained in Reference Example 1 was added to an equal volume of the active bead suspension, and the mixture was incubated at 37 ° C. for 3 hours with slow stirring to bind the capture probe to the beads. (2) Blocking method of capture probe-bound beads After collecting the capture probe-bound beads with a magnet and removing the supernatant,
It was washed twice with sterile water. After washing, the sterilized water was removed, an equal volume of block buffer was added, and the mixture was allowed to stand at 37 ° C. for 3 hours to carry out a block reaction. (3) Preparation of hybridization buffer containing additives Hybridization buffer (5 x SSC, 0.5%
Polyvinylpyrrolidone K-30) with glycoside derivative having the structural formula shown in Table 1 (trade name: SCLAF, Nippon Seika Co., Ltd.)
Was added at a concentration of 1.0% (W / V) to prepare a solution. In addition, as a comparative example, a hybridization buffer containing sodium lauryl sulfate (special reagent for studying poorly soluble protein, manufactured by Nacalai Tesque) as an additive instead of various scrafs was prepared. (4) Detection of Vibrio parahaemolyticus gene by sandwich hybridization method containing various additives in hybridization buffer Using the reagents and samples prepared by the methods of Reference Examples 2 and 3,
The detection of the Vibrio parahaemolyticus gene as a positive sample was performed by the method described below. The sample was denatured for 15 minutes at room temperature by adding an equal volume of 0.6N NaOH. As a control (negative sample), a human placenta-derived DNA (Sigma) denatured by the same method was used. After binding the capture probe, 50 μl of the blocked magnetic bead suspension was dispensed into an Eppendorf tube, the beads were collected with a magnet to remove the block buffer, and 2 μl of the denatured sample and the alkaline phosphatase-labeled probe were included. Then, 100 μl of the hybridization buffer containing the above additives was added, and hybridization was carried out at 50 ° C. for 60 minutes while shaking. After collecting the beads with a magnet, the probe solution was removed and the washing solution (2
× SSC, 1.0% sodium lauryl sulfate (150 μl) was added and washed. Then remove the washing solution and add 1 x SSC to 15
The beads were suspended by adding 0 μl and the whole amount was dispensed into the wells of the microplate. After collecting beads with a magnet and removing 1 × SSC, Lumiph, a substrate of alkaline phosphatase
Add 100 μl of os 480 (manufactured by Wako Pure Chemical Industries, Ltd.), 15 at 37 ℃ in the dark.
A luminescence reaction was performed for a minute. Micro light 1000
(Dynatech). As is clear from Table 3 below, when the reagent of this example is used, the value (background) of the negative sample measured relative to the comparative example is decreased, and the value of the positive sample measured is also increased. , And excellent results were obtained in terms of sensitivity and specificity. On the other hand, it can be seen that sodium lauryl sulfate used as a comparative example not only suppresses nonspecific adsorption, but also inhibits specific reaction.

[0067]

[Table 3]

Example 3 (1) Method for binding capture probe to solid phase A nylon membrane (High Bond N +; Amersham Co.) was used as a solid phase. Membranes were lightly rinsed with 5 × SSC before immobilizing nucleic acids. The Vibrio parahaemolyticus nucleic acid obtained in Reference Example 3 was diluted with sterile water to a concentration of 1 μl / 50 μl. An equal volume of 0.3 N NaOH was added to the nucleic acid aqueous solution and treated at room temperature for 15 minutes to denature it.
After denaturation, 100 μl of each nucleic acid was added onto the nylon membrane using a dot blotter (BRL) and slowly sucked.
After all the solution has been aspirated, add 5 x SSC in 100 μl aliquots, aspirate, remove the membrane from the dot blotter and 5 x
The membrane was rinsed lightly with SSC, and the membrane was drained to dryness. (2) Block of Vibrio parahaemolyticus gene-immobilized membrane and Vibrio parahaemolyticus gene detection method The nucleic acid-immobilized membrane was placed in a hybrid bag (BRL), and a block buffer was added to seal with a policyr, and the mixture was shaken at 50 ° C. Blocked for 15 minutes. The block buffer was removed from the hybrid bag, 100 μl of the hybridization buffer solution containing the above-mentioned additive and containing the alkaline phosphatase-labeled probe was added, and hybridization was carried out at 50 ° C. for 60 minutes while shaking. Remove the membrane from the hybrid bag and wash the solution (2 x SS
C, 1.0% sodium lauryl sulfate) was transferred to a vat and shaken, and washed at 50 ° C. for 10 minutes. Next, it was transferred to a vat of a washing solution (1 × SSC, 0.5% Triton X-100) and washed with shaking at room temperature for 10 minutes. Place the washed membrane in a hybrid bag, add a substrate solution of alkaline phosphatase containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate, seal, and leave at 37 ° C for 1 hour in the dark. It was made to react.
After the color development, the film was washed with distilled water and dried, and the color development was measured using a color difference meter CR-221 (Minolta Camera Co., Ltd.).
ΔL * a * b * mode was used for the measurement. As a comparative example, the membrane on which nucleic acid was immobilized and blocked by the above method was reacted with a hybridization buffer containing an alkaline phosphatase-labeled probe containing sodium lauryl sulfate as an additive, and the color development value was measured after washing. As is clear from Table 4 below, when the reagent of this example is used, the value (background) of the negative sample measured relative to the comparative example is decreased, and the value of the positive sample measured is also increased. Excellent results were obtained in terms of sensitivity and specificity, such as the appearance of On the other hand, it can be seen that sodium lauryl sulfate used as a comparative example not only suppresses nonspecific adsorption, but also inhibits specific reaction.

[0069]

[Table 4]

Example 4 (1) Method for binding capture probe to solid phase As a solid phase, a polystyrene microtiter plate (Microlite 2, Dynatech) was used. The capture probe obtained in Reference Example 1 was diluted with 50 mM H ₃ BO ₃ buffer
Diluted to 0 pico mole / ml. 100 μl of the solution was dispensed into wells of a microtiter plate and incubated overnight at room temperature. (2) Blocking method of capture probe binding plate The capture probe solution was removed from the plate obtained by this method using an aspirator, and the block buffer was added to each well.
Dispense 150 μl each and block at room temperature for 2 hours. (3) Preparation of hybridization washing solution containing additives 1.0% (W / V) of glucoside derivative (trade name: SCLAF, manufactured by Nippon Seika) having the structural formula shown in Table 1 was added to the hybridization washing solution (2 × SSC). A solution added at the concentration of was prepared.

As a comparative example, a hybridization washing solution containing sodium lauryl sulfate (special reagent for researching poorly soluble protein, Nacalai Tesque, Inc.) as an additive instead of various scrafs was prepared.

(4) Detection of Vibrio parahaemolyticus Gene by Sandwich Hybridization Method Including Various Additives in Hybridization Wash Solution Using the reagents and samples prepared by the methods of Reference Examples 2 and 3,
The detection of the Vibrio parahaemolyticus gene as a positive sample was performed by the method described below. Add an equal volume of 0.6N NaOH to the sample at room temperature.
It was denatured for 15 minutes. As a control (negative sample), a human placenta-derived DNA (Sigma) denatured by the same method was used. After binding the capture probe, remove the blocking solution from the blocked plate and add 2 μl of denatured sample.
Was added. Next, hybridization buffer (5 x S
SC, 0.5% Polyvinylpyrrolidone K-30) 100μ
In addition, hybridization was performed at 50 ° C. for 60 minutes. Remove the solution from the well and wash solution (2 x SSC, 1%
200 μl of various additives) were added, and the mixture was washed at 50 ° C. for 5 minutes.
Subsequently, the washing solution was removed, and the well was rinsed with 200 μl of 1 × SSC to prepare an alkaline phosphatase-labeled probe solution.
100 μl was added and hybridization was carried out at 50 ° C. for 60 minutes. Remove the probe solution from the wells and remove the wash solution.
Add 200 μl and wash at 50 ° C for 10 minutes, then wash solution (1
× SSC, 0.5% Triton X-100) (200 μl) was added, and the mixture was washed at room temperature for 10 minutes. Finally, 200 μl of 1 × SSC was added to rinse the well, 100 μl of Lumiphos 480 (Wako Pure Chemical Industries, Ltd.), which is a substrate for alkaline phosphatase, was added, and a luminescence reaction was carried out at 37 ° C. for 15 minutes in the dark. The amount of light emitted is
It was measured with Microlite 1000 (Dynatech). As is clear from Table 5 below, when the reagent of this example was used, the value (background) of the negative sample measured for the comparative example decreased, and the value of the positive sample measured also increased. Excellent results were obtained in terms of sensitivity and specificity, such as the appearance of

[0073]

[Table 5]

Example 5 (1) Method for binding capture probe to solid phase Magnetic beads as a solid phase (Dynal Co .; Dynabeads M-2)
80 Tosyl Activated) was used. Activated beads 1 mM HCl
The suspension was washed once with sterile distilled water. The capture probe obtained in Reference Example 1 was added to an equal volume of the active bead suspension, and the mixture was incubated at 37 ° C. for 3 hours with slow stirring to bind the capture probe to the beads. (2) Blocking method of capture probe-bound beads After collecting the capture probe-bound beads with a magnet and removing the supernatant,
It was washed twice with sterile water. After washing, the sterilized water was removed, an equal volume of block buffer was added, and the mixture was allowed to stand at 37 ° C. for 3 hours to carry out a block reaction. (3) Preparation of hybridization washing solution containing additives 1.0% (W / V) of glycoside derivative (trade name: Clark, Nippon Seika) with the structural formula shown in Table 1 in the hybridization washing solution (2 x SSC). A solution added at the concentration of was prepared. In addition, as a comparative example, a hybridization washing solution containing sodium lauryl sulfate (special reagent for studying poorly soluble protein, manufactured by Nacalai Tesque) as an additive instead of various scrafs was prepared. (4) Detection of Vibrio parahaemolyticus Gene by Sandwich Hybridization Method Including Various Additives in Hybridization Wash Solution Using the reagents and samples prepared by the methods of Reference Examples 2 and 3,
The detection of the Vibrio parahaemolyticus gene as a positive sample was performed by the method described below. The sample was denatured for 15 minutes at room temperature by adding an equal volume of 0.6N NaOH. As a control (negative sample), a human placenta-derived DNA (Sigma) denatured by the same method was used. After binding the capture probe, 50 μl of the blocked magnetic bead suspension was dispensed into an Eppendorf tube, the beads were collected with a magnet to remove the block buffer, and 2 μl of the denatured sample and the alkaline phosphatase-labeled probe were included. 100 μl of hybridization buffer was added, and hybridization was carried out at 50 ° C. for 60 minutes while shaking. After collecting the beads with a magnet, the probe solution was removed, and 150 μl of a washing solution (2 × SSC, 1.0% various additives) was added for washing. Subsequently, the washing solution was removed, 150 μl of 1 × SSC was added to suspend the beads, and the whole amount was dispensed into the wells of the microplate. After collecting beads with a magnet and removing 1 × SSC, 100 μl of Lumiphos 480 (Wako Pure Chemical Industries) which is a substrate of alkaline phosphatase
In addition, a luminescence reaction was carried out at 37 ° C for 15 minutes in the dark. The amount of light emission was measured with Microlight 1000 (Dynatech). As is clear from Table 6 below, when the reagent of this example is used, the value (background) of the negative sample measured relative to the comparative example is decreased, and the value of the positive sample measured is also increased. , And excellent results were obtained in terms of sensitivity and specificity. On the other hand, sodium lauryl sulfate used as a comparative example not only suppresses nonspecific adsorption,
It can be seen that it also inhibits a specific reaction.

[0075]

[Table 6]

Example 6 (1) Method for binding capture probe to solid phase A nylon membrane (Hibond N +; Amersham) was used as a solid phase. Membranes were lightly rinsed with 5 × SSC before immobilizing nucleic acids. The Vibrio parahaemolyticus nucleic acid obtained in Reference Example 3 was diluted with sterile water to a concentration of 1 μl / 50 μl. An equal volume of 0.3 N NaOH was added to the nucleic acid aqueous solution and treated at room temperature for 15 minutes to denature it.
After denaturation, 100 μl of each nucleic acid was added onto the nylon membrane using a dot blotter (BRL) and slowly sucked.
After all the solution has been aspirated, add 5 x SSC in 100 μl aliquots, aspirate, remove the membrane from the dot blotter and 5 x
The membrane was rinsed lightly with SSC, and the membrane was drained to dryness. (2) Block of Vibrio parahaemolyticus gene-immobilized membrane and Vibrio parahaemolyticus gene detection method The nucleic acid-immobilized membrane was placed in a hybrid bag (BRL), and a block buffer was added to seal with a policyr, and the mixture was shaken at 50 ° C. Blocked for 15 minutes. Remove the block buffer from the hybrid bag, add 100 μl of hybridization buffer containing alkaline phosphatase-labeled probe, and shake at 60 ° C at 60 ° C.
Hybridization was performed for a minute. The membrane was taken out of the hybrid bag, transferred to a vat containing a washing solution (2 × SSC, 1.0% various additives), and washed at 50 ° C. for 10 minutes while shaking. Next, wash solution (1 x SSC, 0.5% Tri
tonX-100) bat and shake at room temperature for 1
Washed for 0 minutes. Place the washed membrane in a hybrid bag, add a substrate solution of alkaline phosphatase containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate, seal, and incubate at 37 ° C for 1 hour in the dark. It was made to react. After the color development, the film was washed with distilled water and dried, and the color development was measured using a color difference meter CR-221 (Minolta Camera Co., Ltd.). ΔL * a * b * mode was used for the measurement. As a comparative example, the membrane on which nucleic acid was immobilized and blocked by the above method was reacted with a hybridization buffer containing an alkaline phosphatase-labeled probe containing sodium lauryl sulfate as an additive, and the color development value was measured after washing. As is clear from Table 7 below, when the reagent of this example is used, the value (background) of the negative sample measured is decreased and the value of the positive sample also increased compared to the comparative example. Excellent results were obtained in terms of sensitivity and specificity, such as the appearance of On the other hand, it can be seen that sodium lauryl sulfate used as a comparative example not only suppresses nonspecific adsorption, but also inhibits specific reaction.

[0077]

[Table 7]

[0078]

[Sequence list]

SEQ ID NO: 1 Sequence Length: 26 Sequence Type: Nucleic Acid Topology: Single Strand Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Features Location: 1..26 Method of Characterization: S Other Features: Vibrio parahaemolyticus TDH (Thermostable Direct
Haemolysin) has a sequence homologous to the 339th to 364th nucleotide sequences. Sequence CGGTCATTCT GCTGTGTTCG TAAAAT 26

SEQ ID NO: 2 Sequence length: 24 Sequence type: Nucleic acid topology: Single-stranded sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..24 Method of determining features: S, etc. Features: Vibrio parahaemolyticus TDH (Thermostable Direct
Haemolysin) has a sequence homologous to the 102nd to 125th nucleotide sequences. Array CCCCGGTTCT GAXGAGATAT TGTT 24

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Keisuke Kinomura 5-1-1 Umei, Takasago-shi, Hyogo Prefecture Japan Seika Co., Ltd. (72) Inventor Masakazu Okumura 5-1-1 Umei, Takasago-shi, Hyogo Issue Nippon Seika Co., Ltd. Research Laboratory (56) References J. Biol. Chem. , 1987, Vol. 262, no. 20, p. 9822-9827 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12Q 1/68 CA (STN) REGISTRY (STN) BIOSIS / WPI (DIALOG) JISST file (JOIS)

Claims (2)

(57) [Claims] To 1. A target nucleic acid is a substance to be measured, in nucleic acid hybridization reactions of reacting the nucleic acid complementarily bind, the cleaning system of the reaction system or the reaction, Ok
From chillglucoside and β-octylthioglucoside
A method of nucleic acid hybridization, characterized in that at least one selected is present . To 2. A target nucleic acid is a substance to be measured, in nucleic acid hybridization reactions of reacting the nucleic acid complementarily bind, the cleaning system of the reaction system or the reaction, poly
A method for nucleic acid hybridization, which comprises the presence of glucosyloxyethyl methacrylate .