JPH10108693A - Assay of nucleic acid or the like - Google Patents

Abstract

PROBLEM TO BE SOLVED: To assay nucleic acid or the like in high space resolution power and detection sensitivity by binding a peroxidase to a test specimen such as nucleic acid, protein or microorganisms followed by reaction of a fluorescent substrate with the peroxidase and then conducting an exciting light irradiation to detect the fluorescence emitted. SOLUTION: First, a peroxidase is bound to a test specimen such as nucleic acid, protein or microorganisms followed by reaction of the peroxidase with a fluorescent substrate such as N-phenylcarbonyl-4-amino-2-methoxy-5- methylaniline of formula I,N-phenylcarbonyl-4-amino-2,5-dimethoxyaniline of formula II or N-(4-biphenylcarbonyl)-5-aminofluorescein of formula III. Subsequently, the reaction product is irradiated with exciting light to detect the fluorescence emitted, thus detecting the nucleic acid or the like in high resolution power and sensitivity and in a safe and easy operation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to DNA and RN using fluorescent substrates.
The present invention relates to a method for assaying nucleic acids and proteins such as A.

[0002]

2. Description of the Related Art In the fields of medicine and biology, in recent years, a nucleic acid probe having a specific base sequence has been hybridized with a test nucleic acid having an unknown base sequence.
Determining the nucleobase sequence has been performed. Conventionally, in this method for detecting nucleic acid, for example, a nucleic acid probe is labeled with a radioactive isotope, the nucleic acid is hybridized with a test nucleic acid, and then the radioactivity emitted from the radioactive isotope is measured by autoradiography. In general, an isotope method for detecting nucleic acids is used (Sambrook. J., et.
l. , Molecular Cloning 2nd
edition, Cold SpringHarbor
Laboratory Press. ).

[0003]

However, in the above-mentioned conventional methods for assaying nucleic acids and the like, radioisotopes are used in nucleic acid hybridization to provide a space only for clarifying the relative positional relationship between adjacent genes (between nucleic acid sequences). There is no resolving power. For safety reasons, the experiment must be carried out in an isotope laboratory equipped with special equipment for preventing radioactive leakage. These shortcomings are significant obstacles to the application and development of this field.

[0004] Against this background, in recent years, many non-radioactive labeling methods for DNA and RNA have been developed. For example,
As a typical example, a method for detecting nucleic acid or the like using a chemiluminescent substrate (S. Beck, et. Al., Nuclei
c Acids Res. , 17 (1989) 511
5, I. Bronstein, et. al. , Natu
re, 338 (1989) 599) and a method for detecting a nucleic acid or the like using a chromogenic substrate (J. Magaday, J. His.
tchemie, 23 (1970) 180, J. Am. J. L
early et. al. Proc. Natl. Aca
d. Sci. U. S. A. , 80 (1983) 404
5). These non-radioactive labeling methods are more sensitive than the isotope method. However, the non-radioactive labeling method is as bad as the isotope method in spatial resolution.

The present invention has been made in view of the above-mentioned conventional problems, and has as its object to provide a method for assaying nucleic acids and the like, which has excellent spatial resolution and detection sensitivity and can assay nucleic acids and the like by safe and simple operation. It is.

[0006]

According to the first aspect of the present invention, a nucleic acid, a protein,
Binding peroxidase to a specimen such as a microorganism, and then reacting the peroxidase with a fluorescent substrate to obtain a reaction product, and irradiating the reaction product with excitation light to detect fluorescence generated thereby. This is a method for assaying nucleic acids and the like characterized by the following.

In the method for detecting a nucleic acid or the like according to the present invention, peroxidase is reacted with a fluorescent substrate, and the reaction product is irradiated with excitation light. As a result, the reaction product emits fluorescence. By detecting this fluorescence, the analyte bound to peroxidase can be detected.

That is, when peroxidase reacts with a fluorescent substrate, a reaction product that emits fluorescence upon irradiation with excitation light is generated. For example, as shown in FIG. 1, when the fluorescent substrate is a fluorescein derivative having a fluorescein skeleton (for example, N- (4-biphenylcarbonyl) -5-aminofluorescein), a part of the ring structure of the fluorescein skeleton undergoes hydrolysis. Raise and cleave. This hydrolyzate is a reaction product that can emit fluorescence as described above.
Further, as shown in FIG. 2, an aniline derivative (for example, F
In the case of the first violet B base, the amino groups of two aniline derivatives undergo azo coupling to form an azo compound. This azo compound is a reaction product that can emit fluorescence as described above.

[0009] These reaction products emit strong fluorescence when irradiated with excitation light. Therefore, the detection sensitivity is improved, and a very small amount of the analyte can be detected. For example, when the specimen is DNA, the DNA can be detected down to 0.4 pg or less. Therefore, according to the detection method of the present invention, detection sensitivity superior to the isotope method can be exhibited.

[0010] The above reaction product emits sharp fluorescence when irradiated with excitation light. Therefore, the position of the subject can be clearly imaged by the fluorescent light, and the spatial resolution is excellent. Therefore, the position of the adjacent subject can be clearly shown. For example, when the subject is an electrophoresed DNA band, adjacent DNA bands are displayed as sharp bands, and adjacent DNA bands can be clearly identified.

Further, the fluorescent substrate emits various colors depending on its kind. Therefore, multicolor fluorescence can be obtained by reacting a plurality of types of fluorescent substrates with peroxidase. Peroxidase is an enzyme frequently used in the field of histochemistry. Therefore, it is easily available and easy to handle. Therefore, the detection method of the present invention can be frequently used.

According to the detection method of the present invention, nucleic acids, proteins, microorganisms, and the like can be detected as analytes. However, the present invention is not limited thereto, and any method capable of binding to peroxidase can be used. Can be detected.

Next, as in the second aspect of the present invention, the fluorescent substrate for peroxidase is represented by the following chemical formulas (1) to (1).
It is preferably any one of those shown in "5".
Thereby, higher sensitivity is obtained and spatial resolution is also improved.

[0014]

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[0015]

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[0016]

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[0017]

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[0018]

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[0019]

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[0020]

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[0021]

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[0022]

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[0023]

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[0024]

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[0025]

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[0026]

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[0027]

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[0028]

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Next, as in the third aspect of the present invention, the above-mentioned "Chem. 4", "Chem. 6", "Chem. 7", "Chem. 8", "Chem. 1"
It is preferable that the fluorescent substrate represented by formula (1), (formula 12), (formula 13) or (formula 14) is reacted with the above peroxidase after removing the acetyl group by alkaline hydrolysis. This is because the acetyl group (CH ₃ —CO—) serves as a protecting group for the fluorescent substrate, and thus, by removing the acetyl group from the fluorescent substrate, the reaction between the fluorescent substrate and peroxidase can easily proceed.

Next, the invention of claim 4 relates to nucleic acids, proteins,
A peroxidase for detecting an analyte such as a microorganism, wherein the peroxidase is reacted with a fluorescent substrate in a state of being bound to the analyte to obtain a reaction product, and the reaction product is irradiated with excitation light. A peroxidase characterized in that the analyte is detected by generating fluorescence from a product.

The peroxidase of the present invention is an enzyme that reacts with a fluorescent substrate. Therefore, according to the present invention, as described above, the detection sensitivity of the subject and the spatial resolution can be increased.

[0032]

BEST MODE FOR CARRYING OUT THE INVENTION

First Embodiment An assay method for nucleic acids and the like according to an embodiment of the present invention will be described with reference to FIG. The test method in this example is λDN
This is a method for testing A. First, to the λ DNA of the subject,
After allowing peroxidase to bind and then reacting the peroxidase with a fluorescent substrate to obtain a reaction product, the reaction product is irradiated with excitation light, and the fluorescence generated thereby is detected.

As the fluorescent substrate, those shown in the following samples 1 to 15 were used. N-phenylcarbonyl-4-amino-2-methoxy-5-methylaniline (Fast Vi)
Also called olet B base. ) (Sample 1), N-phenylcarbonyl-4-amino-2,5-dimethoxyaniline (Fast Blue)
Also referred to as RR base. ) (Sample 2), N- (4-biphenylcarbonyl)-
5-aminofluorescein (sample 3), 3 ′, 6′-O-diacetyl-N-
(4-biphenylcarbonyl) -5-aminofluorescein (sample 4), 3′-O- (1-naphthyl) methyl-
N- (4-biphenylcarbonyl) -5-aminofluorescein (sample 5),

3'-O- (3,4-dimethylphenyl) methyl-6'-O-acetyl-N-
(4-biphenylcarbonyl) -5-aminofluorescein (sample 6), 3′-O- (3-indolyl) ethyl-6′-O-acetyl-N- (4-biphenylcarbonyl) -5-aminofluorescein (sample 7), 3'-O- (9-anthranyl) methyl-6'-O-acetyl-N- (4-biphenylcarbonyl) -5-aminofluorescein (shown in Chemical formula 8) Sample 8), 3′-O-acetyl-N- (4-biphenylcarbonyl) -5-aminofluorescein shown in Chemical Formula 9 (Sample 9), N- (3,5- Dimethylphenyl)
-5-thioureidofluorescein (sample 10),

3 ', 6'-O-diacetyl-N- (3,5-dimethylphenyl) -5-thioureidofluorescein (sample 11) shown in Chemical formula 11; 3' shown in Chemical formula 12 —O- (3,4-dimethylphenyl) methyl-6′-O-acetyl-N- (3,5-dimethylphenyl) -5-thioureidofluorescein (sample 12), 3 ′ shown in “Chemical formula 13” -O- (3-indolyl) ethyl-6'-O-acetyl-N- (3,5-dimethylphenyl) -5-thioureidofluorescein (sample 1
3), 3′-O-ethyl-6′-O-acetyl-N- (3,5-dimethylphenyl) -5-thioureidofluorescein as shown in Chemical formula 14 (sample 14), Chemical formula 15 N- (4-methylphenyl) -5-
Thioulaid fluorescein (sample 15) was used.

Samples 1 and 2 were commercially available from Sigma. Samples 3 to 15 were synthesized by the following synthesis method.

(Synthesis Method of Sample 3) First, 202 mg (1.019 mmol) of 4-phenylbenzoic acid was added to 30 ml.
The flask was placed in an eggplant-shaped flask, the flask was sealed, and the air in the flask was replaced with argon gas. Then, under an argon atmosphere, 2 ml of dehydrated and distilled dichloroethane was added, followed by vigorous stirring. Next, a catalytic amount of benzyltriethylammonium chloride was added as a phase transfer catalyst.
Then, 81.4 μl of thionyl chloride (1.121 mm
ol) and heated to reflux for 3 hours to react them. After the reaction, excess thionyl chloride was removed by distillation under reduced pressure.
Then, it was dried under vacuum to obtain 4-biphenylcarbonyl chloride in a yield of 80%.

Next, 176.7 mg (0.815 mmol) of the obtained 4-biphenylcarbonyl chloride was placed in a 10 ml eggplant-shaped flask, and the air in the Clasco was replaced with argon gas. Then, under an argon atmosphere,
1 ml of dehydrated and distilled acetone was added and stirred vigorously, and then 283 mg of 5-aminofluorescein (0.815 m
mol), and the mixture was stirred at room temperature for 12 hours. Next, N- (4
-Biphenylcarbonyl) -5-aminofluorescein (sample 3) was obtained in a yield of 97.2%.

(Method of synthesizing sample 4) First, 100 mg
(0.189 mmol) of the above sample 3 was placed in a 10 ml eggplant-shaped flask, and the air in the flask was replaced with argon gas with a seal cap. Then, under an argon atmosphere, 500 μl of dehydrated and distilled pyridine was added and stirred until dissolved. Next, the reaction solution was cooled to 0 ° C., 39.4 μl of dehydrated and distilled acetic anhydride was added, and the mixture was stirred at 0 ° C. for 2 hours to react them. After the reaction,
By performing reverse phase chromatography, 3 ', 6'-
O-diacetyl-N- (4-biphenylcarbonyl)-
5-Aminofluorescein (sample 4) was obtained with a yield of 92%.

(Synthesis method of sample 5) First, 57.9 mg
(0.11 mmol) of the above sample 3 was placed in a 10 ml eggplant-shaped flask, and the air in the flask was replaced with argon gas with a seal cap. Then, under an argon atmosphere, 400 μl of dehydrated and distilled DMF (meaning dimethylformamide; hereinafter the same) was added, and the mixture was stirred until dissolved. Next, 29.5 mg (0.167 mmol) of 1- (chloromethyl) naphthalene was added, and 1
This was reacted for 0 hour with stirring. After the reaction, 3'-O-
(1-Naphthyl) methyl-N- (4-biphenylcarbonyl) -5-aminofluorescein (sample 5) was obtained in a yield of 9
Obtained at 1%.

(Synthesis method of sample 6) First, 41.0 mg
(0.0777 mmol) of the above sample 3 was placed in a 10-ml eggplant-shaped flask, and the inside of the flask was replaced with argon gas with a seal cap. Then, 400 μl of dehydrated and distilled DMF was added under an argon atmosphere, and the mixture was stirred until dissolved. Subsequently, 20.0 μl (0.137 mmol) of 3,4-dimethylbenzyl chloride was added, and the mixture was stirred at room temperature for 10 hours to be reacted. After the reaction, an intermediate product, 3'-O- (3,4-dimethylphenyl) methyl-5- (4-biphenylcarboxamido) fluorescein, was subjected to reverse phase silica gel column chromatography separation with a yield of 88%. Obtained.

Then, 131.7 mg of this intermediate product
(0.2 mmol) was placed in a 10 ml eggplant-shaped flask, and the air in the flask was replaced with argon gas with a seal cap. Then, under an argon atmosphere, 500 μl of dehydrated and distilled pyridine was added, and these were reacted by stirring until dissolved. The reaction solution was cooled to 0 ° C, 41.4 µl of dehydrated and distilled acetic anhydride was added, and the mixture was stirred at 0 ° C for 2 hours to react. After the reaction, 3′-O- (3,
4-dimethylphenyl) methyl-6'-O-acetyl-
N- (4-biphenylcarbonyl) -5-aminofluorescein (sample 6) was obtained with a yield of 90%.

(Method of synthesizing sample 7) First, 200 mg
(0.379 mmol) of the above sample 3 was placed in a 10 ml eggplant-shaped flask, and the air in the flask was replaced with argon gas with a seal cap. Then, under an argon atmosphere, 1.4 ml of dehydrated and distilled DMF was added and stirred until dissolved. Then, 3- (2-bromoethyl)
Add 102 mg (0.455 mmol) of indole,
These were reacted by stirring at room temperature for 10 hours. After the reaction,
By performing reverse phase silica gel column chromatography,
An intermediate product, 3′-O- (3-indolyl) ethyl-5- (4-biphenylcarboxamido) fluorescein, was obtained in a yield of 78%.

Then, 100 mg of this intermediate product (0.
149 mmol) in a 10 ml eggplant-shaped flask,
The air in the flask was replaced with argon gas with a seal cap. Then, under an argon atmosphere, 500 μl of dehydrated and distilled pyridine was added, and these were reacted by stirring until dissolved. The reaction solution was cooled to 0 ° C., 20 μl of dehydrated and distilled acetic anhydride was added, and the mixture was stirred and reacted at 0 ° C. for 2 hours. After the reaction, 3′-O- (3-indolyl) ethyl-6′-O-acetyl-N- (4-biphenylcarbonyl) -5-aminofluorescein (sample 7) was obtained by reverse-phase silica gel column chromatography.
Was obtained in a yield of 80%.

(Method of synthesizing sample 8) First, 200 mg
(0.379 mmol) of the above sample 3 was placed in a 10 ml eggplant-shaped flask, and the air in the flask was replaced with argon gas with a seal cap. Then, under an argon atmosphere, 1.4 ml of dehydrated and distilled DMF was added and stirred until dissolved. Next, 103.2 mg (0.455 mmol) of 9- (chloromethyl) anthracene was added, and the mixture was stirred at room temperature for 10 hours to be reacted. After the reaction, the intermediate product 3'-O- (9-anthranyl) is separated by reverse phase silica gel column chromatography.
Methyl-5- (4-biphenylcarboxamido) fluorescein was obtained with a yield of 74%.

Then, 46 mg of this intermediate product (0.0
64 mmol) was placed in a 10-ml eggplant-shaped flask, a seal cap was placed, and the air in the flask was replaced with argon gas. Then, under an argon atmosphere, 400 μl of dehydrated and distilled pyridine was added, and these were reacted by stirring until dissolved. The reaction solution was cooled to 0 ° C., 7.3 μl of dehydrated and distilled acetic anhydride was added, and the mixture was stirred and reacted at 0 ° C. for 2 hours. After the reaction, reverse phase silica gel column chromatography was performed to obtain 3'-O- (9-anthranyl) methyl-6'-O-acetyl-N- (4-biphenylcarbonyl) -5-aminofluorescein (sample 8). Was obtained in a yield of 80%.

(Synthesis Method of Sample 9) First, 76 mg
(0.133 mmol) of the sample 3 was placed in a 10-ml eggplant-shaped flask, and the air in the flask was replaced with argon gas with a seal cap. Then, under an argon atmosphere, 300 μl of dehydrated and distilled pyridine was added and stirred until dissolved. Then, the reaction solution was cooled to 0 ° C., and 12.5 μl of dehydrated and distilled acetic anhydride (0.133 μl) was added.
mmol) and stirred at 0 ° C. for 2 hours to react. After the reaction, 3′-O-acetyl-N- (4-biphenylcarbonyl) -5-aminofluorescein (sample 9) was obtained by performing reverse phase silica gel column chromatography separation.
Was obtained in a yield of 70%.

(Synthesis method of sample 10) First, 1 g (2.57 mmol) of 5-fluorene isothiocyanate was added to 1
The flask was placed in a 00 ml eggplant type flask, sealed with a seal cap, and the air in the flask was replaced with argon gas. Then, under argon atmosphere, dehydrated and distilled ethanol 8m
and stirred until dissolved. Next, 330 μl (2.64 mmol) of 3,5-xylysine was added, and the mixture was stirred and reacted at room temperature for 18 hours. After 18 hours, crystals formed and precipitated in the reaction solution. The precipitated crystals were filtered off with suction. The crystals are washed with ether, dried in vacuo,
(3,5-dimethylphenyl) -5-thioureidofluorescein (sample 10) was obtained with a yield of 100%.

(Method for synthesizing sample 11) First, 100 mg
(0.196 mmol) of the above sample 10 was placed in a 10-ml eggplant-shaped flask, a seal cap was placed, and the air in the flask was replaced with argon gas. Then, 400 μl of dehydrated and distilled pyridine was added under an argon atmosphere,
These were reacted with stirring until dissolved. The reaction solution was cooled to 0 ° C., 40.7 μl of dehydrated and distilled acetic anhydride was added, and the mixture was stirred and reacted at 0 ° C. for 2 hours. After the reaction,
By performing reverse phase silica gel column chromatography,
3 ′, 6′-O-diacetyl-N- (3,5-dimethylphenyl) -5-thioureidofluorescein (sample 1
1) was obtained with a yield of 91%.

(Method of synthesizing sample 12) First, 200 mg
(0.402 mmol) of the above sample 10 was placed in a 10-ml eggplant-shaped flask, a seal cap was placed, and the air in the flask was replaced with argon gas. Then, 400 μl of dehydrated and distilled DMF was added under an argon atmosphere, and the mixture was stirred until dissolved. Next, 70.6 μl (0.137 mmol) of 3,4-dimethylbenzyl chloride was added, and the mixture was stirred at room temperature for 10 hours to be reacted. After the reaction, reverse phase silica gel column chromatography was performed. As a result, 3′-O- (3,4-dimethylphenyl) methyl-N- (3,5-dimethylphenyl) -5-thioureidofluorescein as an intermediate product was obtained at a yield of 99%.

Next, 99 mg of the above intermediate product (0.1 mg
6 mmol) was placed in a 10 ml eggplant-shaped flask, and the air in the flask was replaced with argon gas with a seal cap. Then, under an argon atmosphere, 500 μl of dehydrated and distilled pyridine was added and stirred until dissolved. Next, the reaction solution was cooled to 0 ° C., 41.4 μl of dehydrated and distilled acetic anhydride was added, and the mixture was stirred and reacted at the same temperature for 2 hours. After completion of the reaction, reverse phase silica gel column chromatography was performed to obtain 3'-O- (3,4-dimethylphenyl) methyl-6'-O-acetyl-N- (3,5-dimethylphenyl) -5- Thioulide fluorescein (sample 12) was obtained with a yield of 93%.

(Method of synthesizing sample 13) First, 200 mg
(0.402 mmol) of the above sample 10 was placed in a 10-ml eggplant-shaped flask, a seal cap was placed, and the air in the flask was replaced with argon gas. Then, 400 μl of dehydrated and distilled DMF was added under an argon atmosphere, and the mixture was stirred until dissolved. Next, 105.3 mg (0.47 mmol) of 3- (2-bromoethyl) indole was added, and the mixture was reacted by stirring at room temperature for 10 hours. After the reaction, reverse phase silica gel column chromatography was performed. Thus, the intermediate product 3′-O- (3-indolyl) ethyl-N- (3,5-dimethylphenyl) -5-
Thioureid fluorescein was obtained with a yield of 79%.

Next, 68 mg of the above intermediate product (0.1 mg
mmol) was placed in a 10-ml eggplant-shaped flask, and the air in the flask was replaced with argon gas with a seal cap. Then, under an argon atmosphere, 250 μl of dehydrated and distilled pyridine was added and stirred until dissolved. Next, the reaction solution was cooled to 0 ° C., 16 μl of dehydrated and distilled acetic anhydride was added, and the mixture was reacted while stirring at 0 ° C. for 2 hours. After completion of the reaction, reverse phase silica gel column chromatography was performed to obtain 3'-O- (3-indolyl) ethyl-6'-O-acetyl-N- (3,5-dimethylphenyl) -5-thioureidofluorescein. (Sample 13) was obtained with a yield of 79%.

(Method of synthesizing sample 14) First, 200 mg
(0.402 mmol) of the above sample 10 was placed in a 10-ml eggplant-shaped flask, a seal cap was placed, and the air in the flask was replaced with argon gas. Then, under an argon atmosphere, 1 ml of dehydrated and distilled DMF was added and stirred until dissolved. Then, 35.1 μl of bromoethane
(0.471 mmol) was added and stirred at room temperature for 10 hours to react. After the reaction, reverse phase silica gel column chromatography was performed. Thus, the intermediate product 3′-O-ethyl-N- (3,5-dimethylphenyl)
-5-thioureidofluorescein was obtained in a yield of 52%.

Next, 50 mg of the above intermediate product (0.1 mg
mmol) was placed in a 10-ml eggplant-shaped flask, and the air in the flask was replaced with argon gas with a seal cap. Then, under an argon atmosphere, 250 μl of dehydrated and distilled pyridine was added and stirred until dissolved. Next, the reaction solution was cooled to 0 ° C., 9.7 μl of acetic anhydride dehydrated and distilled was added, and the mixture was reacted by stirring at the same temperature for 2 hours. After completion of the reaction, 3′-O-ethyl-6′-O-acetyl-N- (3,5-dimethylphenyl) -5-thioureidofluorescein (sample 14) was obtained by reverse-phase silica gel column chromatography. Yield 32%.

(Synthesis method of sample 15) First, 200 mg (0.514 mmol) of 5-fluorene isothiocyanate was used.
l) was placed in a 10-ml eggplant-shaped flask, and the air in the flask was replaced with argon gas with a seal cap. Then, under an argon atmosphere, 500 μl of dehydrated and distilled ethanol was added and stirred until dissolved. Then, 60.3 μl of ortho-toluidine (0.565 mm
ol) and stirred at room temperature for 18 hours. Crystals formed after about 18 hours and precipitated in the reaction solution. The precipitated crystals were then filtered off with suction, washed with ether and dried in vacuo. As a result, N- (4-methylphenyl) -5-thioureidofluorescein (sample 15) was obtained with a yield of 50%.

In the synthesis methods of Samples 3 to 15, the structures of the intermediate product and the target product were all ¹ H-NM.
It was confirmed by R, IR and MS spectra.

(Detection of λ DNA) Next, λ DNA was detected using the above-mentioned fluorescent substrates (samples 1 and 2). To explain this detection method, first, the DN of Boehringer Manheim is used.
A digoxigenin (antigen)
Λ DNA labeled with the above is used for the herring sperm DNA
Dilute with a dilute solution containing g / ml.

Next, as shown in FIG. 1, this DNA diluted solution 1 was spotted on a membrane 2 made of nitrocellulose. One spot contains 0 pg (picogram), 0.08 pg, 0.4 pg, 2 pg, and 10 pg, respectively.
g of digoxigenin-labeled λ DNA.

Next, this membrane was placed in an oven at 80 ° C. for 30 minutes, and the digoxigenin-labeled λD
NA was immobilized on the membrane. Next, the membrane was immersed in the blocking solution for 30 minutes. Next, peroxidase extracted from horseradish was bound to an anti-digoxigenin antibody to prepare an antibody-labeled peroxidase. When the antibody-labeled peroxidase was dropped onto the membrane, all digoxigenin-labeled λ DNA
A sufficient amount capable of binding to was dissolved in the buffer.

Next, a buffer in which the antibody-labeled peroxidase was dissolved was dropped at the DNA spot site on the membrane. As a result, the above-mentioned antibody-labeled peroxidase was bound to the digoxigenin (antigen) labeling the DNA by an antigen-antibody reaction. Then,
Unreacted antibody-labeled peroxidase was removed by washing with a buffer.

Next, 10 mg of the fluorescent substrate was dissolved in 1 ml of ethanol to prepare an ethanol solution containing 10 mg / ml of the fluorescent substrate. Then, ethanol 450μ
was mixed with 12 ml of a pH 7.2 phosphate buffer containing hydrogen peroxide to prepare a mixed solution. Then,
50 μl of an ethanol solution containing the above fluorescent substrate was added to 12.450 ml of this mixed solution. This gives
A fluorescent substrate solution in which 0.5 mg of the fluorescent substrate (samples 1 and 2) was dissolved was prepared.

This fluorescent substrate solution was dropped on the above-mentioned membrane, and the fluorescent substrate solution permeated the whole membrane.
The permeated fluorescent substrate solution contained 0.5 mg of the fluorescent substrate. Then, the enzyme reaction was carried out by holding the membrane at room temperature for 2 hours.

After the reaction, the excitation light (wavelength 300 to 500 n)
m) was irradiated. As a result, fluorescence was observed from a spot on the membrane. The fluorescence was captured by a CCD camera and printed out by a video printer. As a result, as shown in Table 1, the amount of DNA was 0.4% on the nitrocellulose membrane filter 2.
Regarding the spots of pg to 10 pg, the fluorescence detector 1 was confirmed. Note that 0 pg is a blank test.

[0065]

[Table 1]

Embodiment 2 In this embodiment, peroxidase was detected with the above-mentioned fluorescent substrate (samples 1 to 15).

A method for detecting peroxidase will be described.
First, as shown in FIG. 4, peroxidase 3 extracted from horseradish was put on a membrane 2 made of nitrocellulose.
To 0.01-1 μg (microgram) in terms of protein
Was spotted and air-dried.

Next, a fluorescent substrate solution containing a fluorescent substrate (samples 1 to 15) was prepared. That is, samples 4, 6, 7, 8,
In 11, 12, 13, and 14, the acetyl group in the molecule of each sample was alkali-hydrolyzed by dropping an alkaline solution, and removed from the fluorescent substrate. Thereafter, the mixture was neutralized by adding a phosphate buffer of pH 7.2, and this was used as a fluorescent substrate solution. In addition, other samples 1, 2, 3, 5, 9, 1
Nos. 0 and 15 were neutralized with a phosphate buffer of pH 7.2 to obtain a fluorescent substrate solution.

Next, these fluorescent substrate solutions were added dropwise to the above-mentioned spots on the membrane, and these were added at room temperature for 3 hours.
The enzyme reaction was performed by holding for 0 to 60 minutes. After the reaction, the sample was irradiated with excitation light (wavelength 300 to 500 nm) to generate fluorescence. The results of observing the fluorescence are shown in Table 2.

As shown in the table, the fluorescent substrate of the present invention (samples 1 to 1)
In (5), it can be seen that the reaction proceeds even when 0.01 to 0.1 μg or less of peroxidase is added, and emits fluorescence to the extent that it can be visually observed after irradiation with excitation light.

[0071]

[Table 2]

[0072]

According to the present invention, there can be provided a method for assaying nucleic acids and the like, which has excellent spatial resolution and detection sensitivity and can assay nucleic acids and the like by safe and simple operation.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram illustrating a reaction between peroxidase and a fluorescent substrate (fluorescein derivative) in the present invention.

FIG. 2 is an explanatory diagram illustrating a reaction between a peroxidase and a fluorescent substrate (aniline derivative) in the present invention.

FIG. 3 is an explanatory diagram showing a method for detecting λ DNA in Embodiment 1;

FIG. 4 is an explanatory diagram showing a method for detecting peroxidase in a second embodiment.

[Explanation of symbols]

 1. . . 1. DNA dilution solution, . . Membrane, 3. . . Peroxidase,

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yasumitsu Kondo 5-50-5 Hachigencho, Kariya-shi, Aichi Pref. Aisin Cosmos Research Laboratories Co., Ltd. Chome 50 Aisin Cosmos Research Institute

Claims (4)

[Claims] 1. A peroxidase is bound to an analyte such as a nucleic acid, a protein, a microorganism, or the like, and then a reaction product is obtained by reacting the peroxidase with a fluorescent substrate, and the reaction product is irradiated with excitation light. A method for assaying a nucleic acid or the like, characterized by detecting fluorescence generated thereby. 2. The method according to claim 1, wherein the fluorescent substrate of peroxidase is any one of the following chemical formulas (1) to (15). Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image 3. The method according to claim 2, wherein
"Chemical 6", "Chemical 7", "Chemical 8", "Chemical 11", "Chemical 1"
2. A method for assaying a nucleic acid or the like, wherein the fluorescent substrate represented by Chemical Formula 2 or Chemical Formula 13 is reacted with the above peroxidase after removing an acetyl group by alkaline hydrolysis. 4. A peroxidase for detecting an analyte such as a nucleic acid, a protein, or a microorganism, wherein the peroxidase is reacted with a fluorescent substrate in a state of being bound to the analyte to obtain a reaction product. Thereafter, the subject is detected by generating fluorescence from the reaction product by irradiation with excitation light, thereby detecting the subject.