JPH08256796A - Detection of target nucleic acid and reagent kit therefor - Google Patents

Abstract

PURPOSE: To provide a method for detecting a target nucleic acid capable of reducing background and also capable of highly sensitive and highly specific detection. CONSTITUTION: In this method using e.g. an alkali phosphatase-labeled probe by nucleic acid hybridization process, a color reagent containing 2(4- iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and, as necessary, NaCl and MgCl2 is used; thereby a water-soluble coloring matter is formed to accomplish detection of the target nucleic acid. Therefore, water-soluble coloring matter formation gets possible and there becomes no drawback involving troublesome operations, resulting in background reduction and highly sensitive and highly specific detection of the target nucleic acid.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting a target nucleic acid in a sample by a nucleic acid hybridization method using an enzyme-labeled probe, a color reagent and a reagent kit therefor.

[0002]

2. Description of the Related Art Nucleic acid hybridization reaction utilizes complementation of nucleic acids and is widely used as a highly sensitive reaction technique in the fields of medicine, pharmacy and biochemistry. In addition, since the nucleic acid sandwich hybridization method uses two kinds of nucleic acid probes having a base sequence complementary to the target nucleic acid, the background can be reduced and highly sensitive and highly specific detection can be performed.

The detection of the target nucleic acid by the nucleic acid sandwich hybridization method is carried out by using the label of one of the nucleic acid probes. As the labeling substance of the nucleic acid probe, a radioactive substance, a fluorescent substance, a luminescent substance, a chromogen or its precursor, an enzyme and the like are used. Of these labeling substances, the enzyme has a large turnover number,
It has advantages that it can be detected in a short time, it is highly safe and stable, and the detection signal can be amplified through various substrates.

[0004]

However, in the method of detecting a target nucleic acid by using an enzyme-labeled probe by the nucleic acid hybridization method, the labeled enzyme bound to the target nucleic acid by the enzyme-labeled probe or the labeled enzyme in a free state is used. For detection, there is a method in which a luminescent substance produced by the reaction of a labeled enzyme with a luminescent substrate of the enzyme is measured and detected with a measuring device such as a luminometer. In this method, it is necessary to measure using a special measuring device such as a luminometer. When a labeled enzyme is detected by forming a dye with a color reagent containing a substrate of the enzyme or a substrate and a conjugated enzyme, the dye is usually insoluble, so that the dye is deposited on the membrane carrier and detected qualitatively. Therefore, it is necessary to detect by measuring with a colorimeter, or by solubilizing an insoluble dye and measuring the absorbance of the dye.

When alkaline phosphatase is used as a labeling enzyme, 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as a substrate, nitroblue tetrazolium (NBT) as a chromogen and a buffer are required. Commonly use color reagents including NaCl, MgCl ₂ and ZnCl ₂ . However, this coloring reagent produces an insoluble formazan dye, and in order to detect its color development intensity, the dye is deposited on the membrane carrier and qualitatively detected or measured using a colorimeter. It has the drawback that it must be detected or the insoluble dye must be solubilized with a surfactant or the like before the absorbance of the dye must be measured for detection.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that when alkaline phosphatase is used as a labeling enzyme, 5-
Bromo-4-chloro-3-indolyl phosphate (BCIP), 2- (4-iodophenyl) -3- (4
-Nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium (WST-1) and buffer, optionally NaCl, MgCl ₂ and Zn
The present invention was found to solve these problems by forming a soluble dye by using a coloring reagent containing Cl ₂ .

That is, the present invention is a method for detecting a target nucleic acid in a sample by a nucleic acid hybridization method using an enzyme-labeled probe, wherein a labeling enzyme of the enzyme-labeled probe is used to produce a substrate for the enzyme and a water-soluble dye. A method for detecting a target nucleic acid, which comprises detecting a target nucleic acid by reacting with a coloring reagent containing a chromogen to form a water-soluble dye and measuring the color development intensity thereof.

The present invention is also a color reagent for detecting a target nucleic acid, which contains an enzyme substrate and a chromogen that produces a water-soluble dye.

Furthermore, the present invention provides a plate carrier on which a nucleic acid having a base sequence complementary to the target nucleic acid is immobilized, and an enzyme label is attached to the nucleic acid having the base sequence complementary to the target nucleic acid. A target nucleic acid detection reagent kit comprising a bound labeled probe, a hybridization buffer, a washing solution, and a color reagent for detecting a target nucleic acid containing an enzyme substrate and a chromogen that produces a water-soluble dye.

The present invention is a method for detecting a target nucleic acid in a sample by a nucleic acid hybridization method using an enzyme-labeled probe, preferably a method for detecting a target nucleic acid by a sandwich hybridization method using a plate carrier. is there. The detection method by sandwich hybridization using a plate carrier means that two kinds of nucleic acid probes having a base sequence complementary to a target nucleic acid are prepared, and one of the nucleic acid probes is bound with, for example, a microtiter plate as a solid phase, The target nucleic acid is used as a capture probe (capture probe), and the other nucleic acid probe is labeled with an enzyme to be a detection probe (label probe). First, a capture probe is immobilized on a plate carrier, the target nucleic acid in the sample is denatured if necessary, and then bound to the probe, and then bound to the labeled probe and the bound nucleic acid, and the bound labeled enzyme or free The labeled enzyme in the state of being formed is formed into a soluble dye by a coloring reagent containing a substrate of the enzyme or a substrate and a coupling enzyme,
It is a method of detecting nucleic acid in a sample by measuring the absorbance of the dye. The denaturation operation means, when the target nucleic acid is double-stranded, it is made single-stranded by heat denaturation, alkali denaturation, acid denaturation or the like. The sandwich hybridization may be one step or two steps.

The plate used in the present invention includes, for example, those made of synthetic resin such as polystyrene, polyvinyl chloride, and polycarbonate, and there are high-bonding, medium-bonding, and low-bonding types due to protein binding property. From the viewpoint of nucleic acid binding, a polystyrene high binding type plate is preferable. By using the plate as a solid-phase carrier, it can be used not only as a solid-phase carrier but also as a reaction container during the hybridization reaction, so washing operation by suction is possible and removal of unreacted substances is easy. Is. However, carriers other than plates may be used in the present invention.

As the nucleic acid probe having a complementary strand to the target nucleic acid, an oligonucleotide synthesized by the phosphoamidite method using a DNA synthesizer, a restriction enzyme fragment of plasmid DNA obtained by cloning, or the like can be used. The capture probe can be immobilized on the plate carrier by a covalent bond method, an ionic bond method, an adsorption method, or the like. The labeling method of the enzyme in the labeled probe is a method using a crosslinking agent, a method using a biotinylated probe and an avidinating enzyme,
A method using a digoxigenin-labeled probe and an antibody-binding enzyme, a method using a synthetic oligonucleotide using a linker nucleotide, and the like can be used.

Examples of the labeling enzyme include alkaline phosphatase and the like. As alkaline metal phosphatase, 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) is used as the enzyme substrate.

The detection of the labeling enzyme bound to the target nucleic acid by the labeling probe or the labeling enzyme in the free state is carried out by reacting with a coloring reagent containing a chromogen which produces a soluble dye,
It is a feature of the present invention that a water-soluble dye is formed and its color development intensity is measured. When the enzyme is alkaline phosphatase, the substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and the chromogen tetrazolium dye such as 2- (4-iodophenyl) -3- ( 4-nitrophenyl) -5- (2,4
-Disulfophenyl) -2H-tetrazolium (WS
A nucleic acid in a sample can be detected by producing a water-soluble formazan dye with a color reagent containing T-1) and measuring the absorbance at 450 nm using a plate reader or the like. The coloring mechanism is as shown below.

[0015]

Embedded image

[0016]

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In one embodiment of the present invention, a nucleic acid probe (capture probe) having a base sequence complementary to the target nucleic acid is immobilized on the plate carrier, the target nucleic acid in the sample is bound to the nucleic acid, A nucleic acid (labeled probe) bound with an enzyme label is bound to the bound nucleic acid to form a sandwich hybridization complex, and the labeled enzyme of the labeled probe bound to the target nucleic acid or the enzyme label of the unlabeled labeled probe is labeled with the enzyme. Nucleic acid in a sample is detected by reacting it with a color reagent containing a chromogen that produces a substrate and a water-soluble dye to produce a water-soluble dye, and measuring its color development intensity using an absorptiometer. A chromogen that produces a water-soluble dye is 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,
4-disulfophenyl) -2H-tetrazolium (W
ST-1).

In a more specific embodiment, the labeling enzyme is alkaline phosphatase, the substrate of the enzyme is 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), and a water-soluble dye is produced. The chromogen is 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-
There is a method for detecting a target nucleic acid that is tetrazolium (WST-1).

In another embodiment of the present invention, the enzyme substrate and 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl)
There is a color reagent for detecting a target nucleic acid containing a chromogen that produces a water-soluble dye that is -2H-tetrazolium (WST-1).

In a more specific embodiment, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 2- (4-iodophenyl) -3- (4
There is a color reagent for target nucleic acid detection containing -nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium (WST-1), a buffer and optionally NaCl and MgCl ₂ . The reagent may further contain ZnCl ₂ .

As one embodiment of the present invention, a capture probe having a nucleic acid having a base sequence complementary to a target nucleic acid immobilized on a plate carrier, and a nucleic acid having a base sequence complementary to the target nucleic acid A chromogen that produces a labeled probe bound with an enzyme label, a hybridization buffer, a washing solution, an enzyme substrate, and a water-soluble dye.
(4-iodophenyl) -3- (4-nitrophenyl)
There is a reagent kit for detecting a target nucleic acid which comprises a color reagent for detecting a target nucleic acid containing a water-soluble dye which is -5- (2,4-disulfophenyl) -2H-tetrazolium (WST-1).

[0022]

EXAMPLES The present invention will be described below with reference to examples. Example 1 Detection of Vibrio parahaemolyticus TDH gene [Synthesis of capture probe and labeled probe for TDH gene detection] The capture probe and labeled probe were synthesized by the phosphoamidite method using a DNA synthesizer 391 type (manufactured by Applied Biosystems). . The base sequence of the capture probe is 5'-CGGTCATTCTGCTGTGTTCGTAA.
The base sequence of AAT-3 ', labeled probe is 5'-CCCCGGTTCTGAX
It is AGATATTGTT-3 '. In the sequence, X represents uridine having a linker arm at the 5-position.

[Enzyme Coupling to Labeled Probe] The binding between the synthetic labeled probe and alkaline phosphatase via its linker arm is described in Nucleic Acids Research, 1
4, 6155, 1986.

[Binding of capture probe and solid phase] As the solid phase, a polystyrene microtiter plate (high binding capacity EIA / RIA plate, manufactured by Coaster) is used.
The capture probe obtained by the above method was dispensed in 100 μl aliquots into the wells of the microtiter plate at 25 ° C.
After overnight incubation at room temperature, the capture probe was bound to the plate and then blocked with deoxyribonucleotide triphosphate.

[Preparation of Color Reagent] Alkaline phosphatase substrate solution is 0.2 M Tris-HCl, 0.5
8M NaCl, 1% MgCl ₂ , 0.00136%
ZnCl ₂ , 0.6 mg / ml WST-1, 0.3 m
Consists of g / ml BCIP (pH 8.5). [Sample preparation] TDH-producing Vibrio parahaemolyticus strain and TD
Brain Heart Infusion medium containing non-H producing Vibrio parahaemolyticus strain containing 3% NaCl
Agar) and inoculated at 37 ° C. overnight. Each grown colony was scraped into a 1.5 ml Eppendorf tube and diluted with a dilution buffer (0.1 M NaH ₂ PO ₄ ,
It was suspended in 300 μl of pH 7.0). Furthermore, proteinase K (manufactured by Nacalai Tesque, Inc.) 0.6 mg, lysate (8 M urea, 0.25% sodium dodecyl sulfate,
600 μl of 0.25% sodium lauryl sarcosine, 50 mM ethylenediaminetetraacetic acid, pH 7.6) was added, stirred, and incubated at 60 ° C. for 30 minutes. The resulting lysate was extracted twice with phenol and once with chloroform and then ethanol precipitated to obtain a Vibrio parahaemolyticus nucleic acid sample.

[Detection of Vibrio parahaemolyticus TDH gene] Using the color reagent and the sample prepared by the above method, the detection of Vibrio parahaemolyticus TDH gene was carried out by the method described below.
The Vibrio parahaemolyticus TDH gene was treated with NaOH to denature it. As a control, 1 μg / μl of human placenta-derived DNA (manufactured by Sigma) was denatured by the same method. 2 μl of the denatured sample, 50 μl of hybridization buffer and 50 μl of alkaline phosphatase-labeled probe solution were added to the wells of the plate, and hybridization was carried out at 50 ° C. for 60 minutes. The solution was removed from the wells of the plate, 200 μl of Wash Solution 1 containing 1% sodium lauryl sulfate was added, followed by washing at 50 ° C. for 10 minutes, then 200 μl of Wash Solution 2 containing 0.5% Triton X-100, and the mixture was added at 10 ° C. at room temperature. Wash for 1 minute, then 1 x S
After washing with 200 μl of SC, a color reagent for alkaline phosphatase (0.2 M Tris-HCl, 0.5
8M NaCl, 1% MgCl ₂ , 0.00136% Z
nCl ₂ , 0.6 mg / ml WST-1, 0.3 mg
/ Ml BCIP (pH 8.5)) and add 2 at 37 ° C.
After incubating for an hour, the absorbance at 450 nm (EL-
It was measured with a 310 type microplate auto reader manufactured by Biotech. Table 1 shows the results.

[0027]

[Table 1]

[0028]

INDUSTRIAL APPLICABILITY In the present invention, 2- (4
-Iodophenyl) -3- (4-nitrophenyl) -5
By using-(2,4-disulfophenyl) -2H-tetrazolium (WST-1), it becomes possible to generate a water-soluble dye, and the insoluble dye can be detected qualitatively or a special device can be used. Targets capable of high sensitivity and high specificity detection, eliminating the disadvantages of complicated operations such as measurement and detection using or solubilization and then measurement and detection. A method for detecting nucleic acid can be provided. Further, in the present invention, by using a plate carrier as a carrier by generating a water-soluble dye, the washing operation is easy, and when other carriers such as beads are used, centrifugation and filtration operations are performed. It is also excellent in that it does not require complicated operations.

[0029]

[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 determining features: 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 nucleotide sequence from the 102nd to the 125th. Array CCCCGGTTCT GAATAGATAT TGTT 24

Claims (10)

[Claims] 1. A method for detecting a target nucleic acid in a sample by a nucleic acid hybridization method using an enzyme-labeled probe, wherein the enzyme labeled with the enzyme-labeled probe is used as a chromogen that produces a substrate for the enzyme and a water-soluble dye. A method for detecting a target nucleic acid, which comprises detecting a target nucleic acid by reacting it with a coloring reagent containing a to produce a water-soluble dye and measuring the color development intensity thereof. 2. A nucleic acid hybridization method, wherein a nucleic acid probe (capture probe) having a base sequence complementary to a target nucleic acid is immobilized on a plate carrier, and the target nucleic acid in a sample is bound to the capture probe, Then, a nucleic acid probe (labeled probe) in which an enzyme label is bound to a nucleic acid having a base sequence complementary to the target nucleic acid is bound to the bound nucleic acid to form a sandwich hybridization complex, and the label bound to the target nucleic acid is labeled. The enzyme of the probe or the enzyme of the unlabeled labeled probe is reacted with a substrate of the enzyme and a coloring reagent containing a chromogen that forms a water-soluble dye to form a water-soluble dye, and the intensity of color development is measured. The method for detecting a target nucleic acid according to claim 1, wherein the nucleic acid in the sample is detected thereby. 3. A chromogen that produces a water-soluble dye is 2- (4
-Iodophenyl) -3- (4-nitrophenyl) -5
The method for detecting a target nucleic acid according to claim 1, which is-(2,4-disulfophenyl) -2H-tetrazolium (WST-1). 4. The labeling enzyme is alkaline phosphatase, the substrate of the enzyme is 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), and the chromogen producing a water-soluble dye is 2- (4-iodophenyl)-
The method for detecting a target nucleic acid according to claim 1, which is 3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium (WST-1). 5. A coloring reagent for detecting a target nucleic acid, which comprises an enzyme substrate and a chromogen that produces a water-soluble dye. 6. A chromogen that produces a water-soluble dye is 2- (4
-Iodophenyl) -3- (4-nitrophenyl) -5
The color reagent for detecting a target nucleic acid according to claim 5, which is-(2,4-disulfophenyl) -2H-tetrazolium (WST-1). 7. 5-Bromo-4-chloro-3-indolyl-phosphate (BCIP), 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4).
-Disulfophenyl) -2H-tetrazolium (WS
T-1), buffer and optionally NaCl and Mg
The color reagent for detecting a target nucleic acid according to claim 5, which contains Cl ₂ . 8. A capture probe having a nucleic acid having a base sequence complementary to a target nucleic acid immobilized on a plate carrier, and a label having an enzyme label bound to a nucleic acid having a base sequence complementary to the target nucleic acid. A reagent kit for detecting a target nucleic acid, which comprises a probe, a buffer for hybridization, a washing solution, and a color reagent for detecting a target nucleic acid containing an enzyme substrate and a chromogen that produces a water-soluble dye. 9. A chromogen that produces a water-soluble dye is 2- (4
-Iodophenyl) -3- (4-nitrophenyl) -5
The reagent kit for detecting a target nucleic acid according to claim 8, which is-(2,4-disulfophenyl) -2H-tetrazolium (WST-1). 10. The target nucleic acid coloring reagent is 5-bromo-4-
Chloro-3-indolyl phosphate (BCI
P), 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2
The reagent kit for detecting a target nucleic acid according to claim 8, which contains H-tetrazolium (WST-1), a buffer solution and optionally NaCl and MgCl ₂ .