JPH04148698A - Method for measuring reverse transcriptase using hybridized substance of primer converted into solid phase and template rna - Google Patents

Abstract

PURPOSE:To simply and rapidly carry out measurement of a reverse transcriptase in a test liquid without using a radioactive label and facilitate confirmation of infection, etc., with retroviruses by using a specific method for utilizing a specified template RNA and a primer. CONSTITUTION:A hybridized substance of an artificially prepared adenine ribopolynucleotide template RNA and an oligodeoxythymine nucleotide, converted into the solid phase and complementary to the aforementioned template RNA, is initially reacted with biotin-substituted deoxyuridine triphosphate (biotin-substituted dUTP) in the presence of a test liquid in an aqueous medium. The resultant reaction product is then separated from the unreacted dUTP and the amount of the reaction product or the biotin-substituted dUTP is measured to measure the reverse transcriptase in the test liquid.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a method for determining reverse transcriptase in a test fluid and a method for confirming retrovirus infection in a test subject by measuring reverse transcriptase-neutralizing antibodies in body fluids. Regarding the law.

[Prior Art] Conventionally, the activity of reverse transcriptase has been measured using natural RNA or adenine ribopolynucleotide (hereinafter sometimes abbreviated as polyA) and oligodeoxythymine nucleotide (hereinafter sometimes abbreviated as oligo-dT). ), tritiated deoxythymidine 5''-triphosphate (hereinafter sometimes abbreviated as (3H)dTTP) as a substrate, and oligo-dT using the reverse transcriptase activity in the reaction solution.
A method has been reported in which the elongation is extended, the elongation is filtered through a filter, and the radioactivity in the filter is measured (Dav
idBaltimore Nature, 22
6.1209-1211, (1970); Ho
ward M, Tem1n & SatoshiMi
zutani, Nature, 226.1211-
1213. (1970)).

Furthermore, a method for diagnosing infection with the AIDS virus, which is a type of retrovirus, using the above-mentioned method for measuring reverse transcriptase activity has been reported [JP-A-63-2522
Publication No. 53].

Traditionally, many operations commonly used in biopharmaceutical research and recombinant DNA technology typically involve isotopic hydrogen ("
H), phosphorus (II2P), carbon (“C), iodine (12
It relies heavily on the use of radiolabeled polynucleotides, such as polynucleotides. Such radioactive compounds can then be used to detect, monitor, localize, or isolate nucleic acids or other molecules of scientific interest or clinical interest, even if they are present in extremely small amounts. Since it is possible to
It has been used as a useful indicating probe.

However, the use of radioactive materials is subject to severe restrictions and is associated with serious drawbacks. First of all, the production of radioactive isotopes, the risk of potentially exposing people who work with radioactive materials to high levels of radiation.
There was a problem in that safety measures had to be taken and maintained during use and post-processing.

Second, radioactive nucleotides are expensive, which increases the cost for general use.
Third, to obtain the necessary sensitivity, radioactive materials with high specific activities must be used, but radioactive materials with high specific activities have correspondingly short half-lives and limited shelf lives. There was a problem in that it imposed restrictions on its use.

Therefore, methods using non-radioactive labeling substances in place of radioactive substances are being considered. When the reaction was carried out using deoxyuridine 5'-triphosphate (hereinafter sometimes abbreviated as biotinylated dUTP), biotinylated dUTP was not used as a substrate at all, no elongation of oligo-dT was observed, and reverse transcriptase It has been reported that it cannot be used to measure the activity of [Penn1n
a R, Langer, Alex A, Waldr.
op & David C, Ward Proc, Na
tl, Acad, Sci, usA.

78, 6633-6637. (1981)], biotinylated nucleotides were considered to be inapplicable to measuring the activity of reverse transcriptase synthase.

The present inventors previously conducted extensive research to obtain a measurement system for reverse transcriptase activity that does not use radioactive isotopes, and found that if a specific template DNA and primer were used, biotinylation could occur in the presence of reverse transcriptase. It has been found that dUTP can be used as a substrate and reverse transcriptase activity can be easily measured.

[Problems to be Solved by the Invention] However, the above method still has some problems, and improvements have been sought. In other words, the above method has the problem that the operating procedure is complicated and it takes some time after the reaction starts until measurement can be performed. was required to be established.

[Means for Solving the Problems] As a result of further research in view of the above problems, the present inventors found that the above problems can be solved by using a solid-phase brimer, which is simple and quick. They discovered that reverse transcriptase activity can be measured and completed the present invention.

That is, the present invention at least hybridizes an artificially prepared adenine ribopolynucleotide template RNA and an immobilized oligodeoxythymine nucleotide complementary to the adenine ribopolynucleotide template RNA in an aqueous medium. The hybridized product and biotinylated deoxyuridine triphosphate are reacted in the presence of a test solution, and after separating the reaction product of the reaction and unreacted biotinylated deoxyuridine triphosphate, the reaction product or unreacted 1. A method for determining reverse transcriptase in a test solution, which comprises measuring the amount of biotinylated deoxyuridine triphosphate.

The present invention also provides at least hybridization of an artificially prepared adenine ribopolynucleotide template RNA and an immobilized oligodeoxythymine nucleotide complementary to the adenine ribopolynucleotide template RNA in an aqueous medium. The hybridized product and biotinylated deoxyuridine triphosphate are reacted in the presence of a sample body fluid and reverse transcriptase, and the reaction product and unreacted biotinylated deoxyuridine triphosphate are separated. A method for confirming retrovirus infection in a subject, characterized by measuring the amount of biotinylated deoxyuridine triphosphate or unreacted biotinylated deoxyuridine triphosphate, and determining the reverse transcriptase neutralizing antibody titer in the subject's body fluid from this amount. be.

In the present invention, the RNA used as a template is an artificially prepared adenine ribopolynucleotide (
Hereinafter, such RNA may be abbreviated as polyA).

Poly A is, for example, artificially prepared by synthesis using genetic engineering technology or chemical synthesis technology, and a particularly preferred embodiment is poly A consisting of 100 to 500 bases, which can usually be used advantageously. An example of this is polyA having an average chain length of about 255 bases.

The immobilized oligodeoxythymine nucleotide (hereinafter sometimes abbreviated as immobilized oligo-dT) acts as a primer, and a preferred embodiment is a deoxythymidine oligomer of 11 or more bases, and the longer the Although preferred, what is exemplified as something that can be used advantageously is solid-phase oligo dT having an average chain length of about 15 bases to 21 bases.

This immobilized oligo dT is obtained by immobilizing oligodeoxythymine nucleotides (oligo dT) in the same phase.

Examples of the same phase include, but are not limited to, natural or synthetic polymeric carriers such as glass particles, cellulose, and polystyrene.

Among these, examples of synthetic polymer carriers include styrene and polystyrene, which is a homopolymer or copolymer of styrene (hereinafter referred to as styrene derivative monomer) having a sulfonic acid group, carboxyl group, or amino group as a substituent. and styrene or styrene derivative monomers and methylstyrene, ethylstyrene, chlorostyrene, ethylene, propylene, acrylic acid, acrylic acid methyl ester, acrylic acid ethyl ester, methacrylic acid, methacrylic acid methyl ester, methacrylic acid ethyl ester,
Acrylonitrile, acrylamide, maleic acid, fumaric acid, butadiene, chlorprene, isoprene, vinyl chloride, vinylidene chloride, vinyl acetate, vinyltoluene,
Examples include copolymers with divinylbenzene, polyvinyltoluene, polyester, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polyvinylpyrrolidone, polyvinyl acetate acrylate, vinyl chloride-acrylate, and the like. Further, the surface of this synthetic polymer carrier may be pretreated with a nonionic surfactant.

Particularly preferred examples include polymers or copolymers of styrene, chlorostyrene, acrylic acid, vinyltoluene, and methacrylic acid methyl ester.

As a method for immobilizing oligo dT on these solid phases,
Although both the adsorption method and the chemical bonding method can be used, the chemical bonding method is preferable because higher sensitivity can be obtained.

Particularly preferred is a method in which the 5' end of oligo-dT is chemically bonded to a solid phase. For example, oligo-dT is bonded to the amino group of a solid phase that has or has introduced an amino group, such as polystyrene or polypeptide, which has an amino group. A method of activating the phosphorus #!
-dimethylaminopropyl)carbodiimide hydrochloride or the like, preferably by adding 1-methylimidazole.

In addition, as another method, for example, the above-mentioned condensing agent is applied to the phosphoric acid residue at the 5' end of oligo dT to the carboxyl group of a solid phase having or introducing a carboxyl group such as styrene having a carboxyl group. Examples include a method in which an amino group is bonded in advance and then a carboxyl group and an amino group are subjected to dehydration condensation.

Furthermore, solid phases having or having functional groups such as sulfonic acid groups, hydroxyl groups, dimethylamino groups, etc.
The 5° end of T or oligo dT is made into a suitable functional group,
Both may be combined using a suitable spacer.

The shape is not particularly limited, and may be solidified on the surface of a ball, particulate, or fiber, or on the wall of a plate, container, or the like.

The amount of immobilized oligo-dT bound to the solid phase per unit area is preferably 1.2 pmol/Cm2 or more, and the highest absorbance is usually achieved up to a binding amount of 5.0 pmol/Cm2. It is possible to measure the binding amount even if the binding amount is higher than that, but if the binding amount is too large, it is unfavorable from the viewpoint of sensitivity and economic efficiency, and the binding amount is preferably 1.2 to 5.0.
A range of pmol/cm2 is exemplified.

Biotinylated deoxynucleoside triphosphate (hereinafter sometimes abbreviated as biotinylated dUTP or labeled substrate) used as a substrate is, for example, a compound in which a biotin group and a uracil deoxynucleoside group are bonded via a suitable spacer such as an arylamine. For example, 5-(N-
(N-biotinyl-ε-aminocaproyl)-3-aminoaryl]-deoxyuridine triphosphate (
piotinylated-11-dUTP), 5-(N-[N-(
The biotinyl group is converted into dUTP via an appropriate spacer, such as biotinyl-ε-aminocaproyl)-γ-aminobutyryl-3-aminoaryl-deoxyuridine triphosphate (biotinylated-16-dUTP).
It is a compound combined with Biotinylated-11-dUT
P and piotinated-16-dUTP are commercially available compounds, and other compounds are also available from JP-A-57-
It is easily prepared according to the method described in known literature such as No. 209297.

As the reagents and compositions thereof used for measuring the amount of biotin, those generally used for measuring the amount of biotin can be employed, such as FITC (fluoreracene isothiocyanate) fluorescence detection composition, par Oxidase detection composition, acid phosphatase detection composition, alkaline phosphatase detection composition, β-
Examples include compositions for detecting galactosidase.

The composition for FITC fluorescence detection includes, for example, rabbit IgG.
Compositions consisting of biotin antibodies and FITC-conjugated goat anti-rabbit IgG antibodies, FITC-conjugated avidin, etc. may be mentioned, and peroxidase detection compositions include, for example, streptavidin-biotinylated horseradish peroxidase complex, phosphate Ili. Examples include a suitable buffer such as a fresh solution and a composition consisting of orthophenylenediamine and hydrogen peroxide. Further, as a composition for detecting acid phosphatase, for example, a streptavidin-biotinylated acid phosphatase complex, a suitable fresh solution such as a phosphate buffer, a chromogenic substrate such as paranitrophenyl phosphate, or 4-methylumbelliferyl phosphate can be used. Examples of alkaline phosphatase detection compositions include streptavidin-biotinylated alkaline phosphatase complexes, appropriate yi buffer solutions such as Tris-HCl buffer, and para-nitrophenyl phosphate. A composition comprising a chromogenic substrate or a fluorescent substrate such as 4-methylumbelliferyl phosphate, streptavidinated alkaline phosphatase, Tris-
Suitable solution such as hydrochloric acid buffer! ! A composition comprising a chromophore and a chromogenic substrate such as para-nitrophenyl phosphate or a fluorescent substrate such as 4-methylumbelliferyl phosphate can also be used as a composition for detecting β-galactosidase.
For example, a streptavidin-β-galactosidase conjugate, an appropriate buffer such as a phosphate buffer, and a chromogenic substrate such as 2-nitrophenyl-β-galactoside or 4-
Examples include compositions comprising fluorescent substrates such as methylumbelliferyl-β-galactoside.

These compositions may be those already commercially available, or may be prepared in various ways with reference to known literature and used as reagent compositions for measuring the amount of biotin.

To carry out the method of determining reverse transcriptase in a test solution of the present invention, first, poly A and immobilized oligo dT are hybridized, and then a hybridized product prepared by thorough washing is used, The hybridized product and biotinylation d
The amount of biotinylated dUTP may be measured after reacting with UTP in the presence of a test solution and removing unreacted biotinylated dUTP.

Poly A (average chain length: 255 bases) and immobilized oligo dT (
Average chain length: 21.5 bases) The quantitative relationship is that when solid-phase oligo dT is 1 (molar ratio), polyA is 3 to 0.
It is preferable that the ratio is 0.07, and if appropriate, the solid phase oligo d
PolyA preferred in terms of binding amount per unit surface area of T
The amount should be .

Hybridization is carried out in an aqueous medium such as various buffer solutions or physiological saline adjusted to a pH of 4 to 9, preferably 5 to 8, but salts etc. may be added to this, and hybridization may be inhibited. An organic solvent such as formamide may be added unless necessary. Furthermore, the temperature of the various electrolytes in the aqueous medium is preferably higher in terms of facilitating hybridization.

In addition, the processing time required for hybridization is usually 1~
10 for sufficient hybridization for 24 hours.
The hybridization temperature is usually above the freezing temperature and about 50°C or less, preferably about 4 to 42
It is said to be around ℃.

Aqueous media such as various buffer solutions and physiological saline are used to wash the hybridized product, and the electrolyte concentration thereof is preferably the same as or higher than the concentration of the subsequent reverse transcriptase measurement solution. In the present invention, since a solid-phase brimer is used, the hybridized product can be washed by filtration,
This can be easily carried out by centrifugation, decantation, etc., and the number of times of washing is usually about 3 to 5 times, and it is sufficient if the amount and number of washings are sufficient.

The reaction consists of a test solution, the polyA-immobilized oligo dT hybridized product, and biotinylated dtJT as a labeled substrate.
Tris-HCl buffer adjusted to pH 6.5 to 8;
0.5 at around 37°C in vIi sanitary fluid such as phosphoric acid deconcentration solution
After incubation for ~24 hours, the biotinylated dUTP is bound to a portion of the primer of the polyA-immobilized oligo-dT hybridized product by the action of reverse transcriptase present in the system.

The test solution may be any test solution for measuring the presence or absence of reverse transcriptase or the enzyme activity of reverse transcriptase, such as a culture solution of retrovirus-infected cells.

Furthermore, the biotinylated dUTP bound to the polyA-immobilized oligo dT hybridized product (hereinafter sometimes referred to as the reaction product) and the unreacted biotinylated dUTP can be separated using immobilized oligo dT. Therefore, it can be easily carried out by filtration, centrifugation, decantation, etc.

The amount of biotinylated deoxyuridine used as a substrate, that is, the amount of reacted piotinylated dUTP, can be determined by measuring the amount of biotin in biotinylated deoxyuridine bound to the primer through the elongation step using a reagent for measuring biotin. For example, When a composition consisting of streptavidinated alkaline phosphatase, Tris-HCl buffer, and para-nitrophenyl phosphate is used as a reagent for measuring biotin content, it is carried out as follows.

i.e. pH 7 containing 0.15-0.5M NaCl
, 0.05 to 0.05 to Tris-HCl buffer adjusted to 5 to 9.5.
10,ug/mH! The solid phase carrier on which the biotinylated dUTP has been reacted is immersed in a solution obtained by adding streptavidinated alkaline phosphatase for about 10 to 60 minutes. Wash the in-phase carrier at least three times with a sufficient amount of the above l1m solution, and then wash it with, for example, 50mM diethanolamine-hydrochloric acid solution containing 1mM MgC12 (p1m solution).
A solid phase carrier was added to a solution obtained by dissolving 1 mg/ml paranitrophenyl phosphate in H9,5-10).
．． Soak for 1 to 2 hours to develop color. The amount of color developed can be determined by measuring the absorbance at a wavelength of 405 nm.

The amount of biotinylated dUTP bound thus measured reflects the amount of reverse transcriptase in the system, that is, the amount of reverse transcriptase in the test solution, so the amount of reverse transcriptase, etc. can be determined from this. .

Furthermore, in addition to labeled substrates, to further increase detection sensitivity,
Deoxythymidine triphosphate (hereinafter referred to as
(sometimes abbreviated as dTTP) can be added. The amount of dTTPO used for this purpose is usually 2 to 20 times, preferably 3 to 4 times, in molar ratio compared to the amount of biotinylated dUTP, which is a labeled substrate.

The amount of reverse transcriptase determined by the method of the present application is usually 0.000.
A reverse transcriptase of about 0.05 to 1 unit can be determined with good sensitivity.

Furthermore, when carrying out the method of the present invention for confirming retrovirus infection in a subject by detecting reverse transcriptase-neutralizing antibodies in body fluids, at least biotinylated dUTP is used as a substrate in the presence of the subject body fluid and reverse transcriptase, This is reacted with a hybridized product of polynucleotide and immobilized oligo-dT. In this step, a reverse transcriptase-containing solution whose enzyme activity has been measured in advance is usually 0.001 to 1 unit, preferably 0.001 unit.
~0.01 unit, mixed with the sample body fluid, and heated at about 4°C for usually 0.1 to 2 hours, preferably 10
After incubation for ~60 minutes, an 11-sided solution such as 12 m5 of Tris-salt adjusted to pH 6.5-8 containing biotinylated dUTP as a substrate, usually 0.5-100 μM, preferably 1-5 μM, is added. PolyA present
and immobilized oligo dT to the hybridized product, and incubated at about 37°C for about 0.5 to 24 hours.

Reverse transcriptases include human immunodeficiency virus type 1 (H), human immunodeficiency virus type 2, rhesus monkey immunodeficiency virus, mouse mammary adenoma virus, human T-cell leukemia virus type D, Rous strain avian sarcoma virus, Rous strain. Viral particles such as retroviruses such as related viruses type 2 and hepatitis B virus (HBV) are treated with nonionic surfactants, such as polyoxyethylene alkylphenyl ether (
Triton X-100, NP-40) can be used.

The body fluid to be tested may be any body fluid contained or produced in the human body that has antibodies against reverse transcriptase, preferably serum.

Serum that has been precipitated with 30 to 50% saturated ammonium sulfate, or that has been passed through a cation exchange resin such as DEAE or QAE-Sephadex, is usually used.

The method for separating reacted and unreacted biotinylated dUTP and measuring their amounts may also be in accordance with the method for determining reverse transcriptase described above.

Thus, reacted or unreacted piotinylated dUT in the measurement system.
Although the amount of P is determined, the reverse transcriptase in the system is inactivated by antibodies derived from the body fluid to be tested, and as a result, the amount of reactive piotinated dUTP decreases. Therefore, by using positive body fluid control and negative body fluid control as indicators, it is possible to measure the reverse transcriptase neutralizing antibody titer in the body fluid, and this antibody titer indicates the retrovirus infection of the subject who has the body fluid. It is an indicator of presence/absence and recognition.

In the present invention, the quantitative relationship between poly A (average chain length: 255 bases) and immobilized oligo dT (average chain length: 21.5 bases) is based on the assumption that the immobilized oligo dT is 1 (molar ratio). In some cases, it is preferable to set the ratio of poly A to 3 to 0.07, and the amount of poly A may be set appropriately based on the amount of bonding per unit surface area of the solid-phase oligo dT.

Furthermore, in order to further increase the detection sensitivity, dTTP can be added as a substrate in addition to the labeled substrate. The amount of dTTP used for this purpose is usually 2 times to 2 times the amount of pyotinated dUTP, which is the labeling substrate, in terms of molar ratio.
0 times, preferably 3 to 4 times.

In order to carry out the method of the present invention advantageously, for example: (1) a hybridized product of artificially prepared adenine ribopolynucleotide template RNA and immobilized oligodeoxythymine nucleotide complementary to the template RNA; (2) a pyotinated deoxyuridine trinucleotide; A kit for measuring reverse transcriptase can be used, which contains phosphate and (1) a reagent for measuring the amount of piotin.

These kits may be in a solution state, but they may also be in the form of lyophilized reagents that are dissolved in deionized water, physiological saline, various buffers, etc. at the time of use.

[Effects of the Invention] According to the method of the present invention, it has become possible to detect, for example, reverse transcriptase activity in the lymphocyte culture medium of an HIV carrier without using a radioactive label. In addition, it has become possible to measure the amount of anti-transferase-neutralizing antibodies, which are closely related to the development of AIDS, without using radioactive substances, making it possible to easily monitor the possibility of the onset of HIV in HIV carriers.

In particular, the method of the present invention uses immobilized oligo dT and polyA-immobilized oligo dT hybridized product, so the reaction time is short and separation of reaction products and unreacted substances is extremely easy. This makes it possible to shorten measurement time and process a large number of samples, which is convenient for mechanization.

Also, oligo dT is 1 (molar ratio) and poly A is 0.0.
When the ratio is 7 to 3, the sensitivity is good and the polyAtt
When O.5 to 2.6, it became possible to measure even more preferably.

[Examples] Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Comparison of reaction rates between a reverse transcriptase reaction using a hybridized product of pre-prepared template polyA and immobilized oligo dT12-+s and a liquid phase reverse transcriptase reaction: (1) Oligo dT,... ! 100 mM 1-C tyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (abbreviated as CDI; Peptide Institute theory) and 100 mM 1-methyl-imidazole-hydrochloric acid buffer (pH 7, 0, abbreviated as IMD; SI
Oligo dT+2-+s in an aqueous solution containing
(Average chain length: 15 bases, average molecular weight: 4,950 Daltons; manufactured by Pharmacia) 200 ng 750 μl
Dissolve 50 μm of the solution into wells of an amino plate (bottom area: 0.32 cm; manufactured by Juman Bakelite Co., Ltd.) (the surface area of the wells in contact with the reaction solution is 0.0 μm).
63 cm') and allowed to react at room temperature for 24 hours. After the reaction, discard this solution and add 0.1
M Tris-HCl buffer (pH 7.5; hereinafter referred to as TBS)
Wash the wells twice with 200 μm of
Wash the wells with 200 μM buffer and add twice with TBS.
It was washed at 200 μm and used as a solid-phase primer (oligo dT+2-to immobilized plate).

(2) Hybridization of template polyA and immobilized primer 0.1M Tris-HCl buffer containing 0.5M NaCl (
100 at pH 7.5 (hereinafter referred to as high salt concentration TBS)
ng/50 μl of polyA (average chain length: 255 bases, average molecular weight: 86,700 daltons; manufactured by Pharmacia) was added to the oligo dT+2-+e fixed plate prepared in (1) above. , 4°C
After hybridization for 18 hours with high salt concentration TBS
Washed 5 times.

(3) Reverse transcriptase reaction To carry out the DNA chain elongation reaction by reverse transcriptase,
2 foil-made reaction slow ff1li solution (100mM Tris-HCl buffer (pH 7,8), 24mM dithiothreitol, 2.4mM reduced glutathione, 20mM
k (g C12, 320mM KCI, 2mM x tyrene glycoltetraacetic acid, 0.4% Triton X-100,
0.4 nmol of biotinylated-11-dUTP (manufactured by Enzo) was added to 50 μm of a buffer solution consisting of 4% ethylene gelcol, and this was immobilized as described in (2) above. It was fractionated into the well where Brimer and polyA were hybridized. Next is phosphoric acid! ! Sanitary saline (
15 mU150 at pH 7.4 (hereinafter referred to as PBS)
Reverse transcriptase (Rouss-related virus type 2) prepared in μl
RAV-2) derived from Takara Shuzo) was added to a 50 μm well, mixed, and then allowed to act at 37° C. for 1.2.3 and 24 hours.

(4) Quantification of enzyme reaction product (biotinylated DNA) 10 μm of 5M NaCl was added to each reaction solution obtained as described in (3) above, and after release for 5 minutes,
0.5M NaCl and 0.05M MgCl°.
The wells were washed with 200 μm of 1M Tris-HCl mfi solution (pH 9.5, hereinafter referred to as washing solution), and washed 2 to 4 times with 200 μl of the washing solution.

Then 10% of 3% bovine serum albumin dissolved in the washing solution
0 μl was dispensed into wells and incubated at 37°C for 1 hour. After discarding the 3% bovine serum albumin solution, add alkaline phosphatase-labeled streptavidin diluted 1000 times with the washing solution (final concentration 0.088 μg/m1).
, manufactured by BRL) into wells, and incubated at room temperature for 50 μm.
Incubated for hours.

After removing excess alkaline phosphatase-labeled streptavidin by washing the wells 3 to 5 times with a 200 μm washing solution, biotinylated DNA can be quantified by measuring the enzymatic activity of alkaline phosphatase remaining in the wells. And so.

Measurement of the enzymatic activity of alkaline phosphatase was performed by adding paranitrophenyl phosphate to 50 mM jetanolamine-hydrochloric acid buffer (p
H9,5) to a concentration of 1 mg/m1, 50 μl of the solution was added to each well, incubated at 37°C for 30 minutes, and then 50 μl of 0.5N NaOH was added to each well.
The enzymatic reaction was terminated by adding m to the reaction solution, and the absorbance at an absorption wavelength of 405 nm, which was colored by alkaline phosphatase activity, was measured using a plate reader (J-200
The measurement was carried out using a model 0 (manufactured by Intermed).

Table 1 shows the absorbance obtained when reacting for 1 hour, 2 hours, 3 hours, and 24 hours using 15 mU of reverse transcriptase.

Table 1 Reaction time (h) Absorbance 1.0 2 5 1.938 2.2 3 3 2.7 1 2 As shown by the above results, the elongated chain reaction was time dependent. In this measurement method, it is known that reliable measurement values can be obtained at absorbances of 0.1 or higher, so according to the method of the present invention, quantitative determination can be performed already one hour after the start of the reaction. is understood. On the other hand, in the comparative method of Reference Example 1 (reaction in a liquid phase system) shown below, measurements could be easily made 2 hours after the start of the reaction.

As described above, the method of the present invention, which uses immobilized primers to perform a reverse transcriptase reaction, is superior in that it can measure reverse transcriptase activity in a shorter time than when reaction is performed in a liquid phase system. It is clear that

Reference Example 1 Change over time of reverse transcriptase reaction product in a liquid phase system Reverse transcriptase reaction 1 with 2 confidence levels described in Example 1 (3)
Add 1 μg of oligo dT+2−+s to 50 μm of buffer solution, 0.
1 μg polyA, 0.4 nmol biotinylated-11-
dUTP was added, and 15 mU of RAV-2-derived reverse transcriptase was added thereto to give a total amount of 100 μm, and the mixture was allowed to act at 37°C for 1, 2, 3 and 24 hours.

A solution containing 10 times the amount of IN hydrochloric acid and 0.1 M sodium pyrophosphate was added to this reaction solution, and after stirring and mixing, 1
Cooled on ice for 0 minutes.

This solution is suctioned and filtered through a 96-hole manifold (manufactured by BRL) equipped with a nylon membrane 100C 2 (manufactured by Nippon Ball Co., Ltd., trade name: Biodyne), and the elongated biotinylated DNA strands are transferred to the nylon membrane. I was able to absorb it.

To the nylon membrane adsorbing the biotinylated DNA obtained here, 3% BSA was added to buffer solution 1 (a buffer solution consisting of 0.1M Tris-HCl I! fresh solution pH 7.5) and 0.15M NaCl). and incubated at 65°C for 1 hour.

The nylon membrane adsorbed with biotinylated DNA was taken out from the solution, and then placed in an alkaline phosphatase I-recognizing streptavidin solution diluted 1,000 times with buffer 1 for 10 minutes at a ratio of 7 ml of the solution per 100 cm of nylon membrane. . Subsequently, the nylon membrane was exposed to 1,100 ml of buffer solution for 15 minutes, and the nylon membrane, which had also adsorbed biotinylated DNAg twice, was washed by exposing it to 100 ml of buffer solution 1 for 15 minutes.

Furthermore, the nylon membrane that had adsorbed the piotinated DNA chain was taken out, and buffer solution 2 (0.1M Tris-mMfiffi
solution (pH 9,5), 0.1M NaCl and 50 m
1 in 100 ml (buffer consisting of M M g Cl 2)
Washed for 0 minutes.

The color was developed using 0.33 mg/ml of nitro blue tetrazolium (NET) and 0.17 mg/ml of 5-
Bromo-4-chloro-3-indole-phosphate (BC
IP) containing buffer solution 2 (7 per 100 cm of nylon membrane)
．． (used at a rate of 5 ml) for 30 minutes. The colored spots that appeared on the nylon film were measured using a densitometer (C8-930, manufactured by Shimabara Seisakusho) at a wavelength of 550 nm.
Measured in m.

1.2.3 and 24 of reverse transcriptase reaction in liquid phase system
The areal intensities of the reaction products over time are shown in Table 2.

Table Reaction time Area intensity 2.3 7.3 9.2 Under these measurement conditions, reliable measurement is possible when the area intensity is 7 x 103 or more.

Therefore, in the liquid-phase reverse transcriptase reaction, sufficient quantitative areal intensity was not obtained 1 hour after the start of the reaction, but quantitative areal intensity was obtained from 2 hours after the start of the reaction.

Example 2 Reverse transcriptase reaction in hybridized products of pre-prepared template polyA and immobilized dT+o-2□ Oligo dT l-24 (average chain length: 21 According to the method of Example 1 (2), 10 μg, 1 μg, 1100 n, Long and ln were added to the solid phased brimer immobilized with 0.5 base, average molecular weight near 095 daltons; manufactured by Pharmacia.
A hybridized product was prepared by hybridizing poly A of g and washing. To this hybridized product, 0.96 nmol of dTT was added to the double concentration foil transfer enzyme buffer containing piotinylated-11-dUTP described in Example 1 (3).
50 μm of a mixed solution containing P (manufactured by Boehringer Mannheim) was added, and 0.1 mTJ of RAV-2-derived reverse transcriptase was further allowed to act at 37° C. for 18 hours. Thereafter, the biotinylated DNA extended on the solid phase was measured using the method described in Example 1 (4).

Reference Example 2 Reverse transcriptase reaction when poly A was added at the start of the reverse transcriptase reaction without hybridization in advance.
0.24 nmol of piotinylated-11-dU in 0 μl
TP and 0.96 nmol dTTP, 10 μg,
1 μg, 1100n, 10ng and lng of each member of polyA were added, and this was added to the oligo d prepared in Example 2.
It was aliquoted into each well of a Tlo-2a immobilized plate. Next, 50 μl of RAV-2 reverse transcriptase, prepared in the same manner as 0.1 mU 150 μl in PBS, was added to the well, mixed, and incubated at 37° C. for 18 hours to allow the reverse transcriptase to act. Thereafter, the biotinylated DNA extended on the solid phase was measured using the method described in Example 1 (4).

In the case where the immobilized primer and each member of poly A were hybridized in advance (Example 2) and the case where each amount of poly A was added at the start of the reverse transcriptase reaction (Reference Example 2), 0.
Table 3 shows the absorbance obtained when 1 mU of RAV-2-derived reverse transcriptase was allowed to act at 37°C for 18 hours.

Table 3 (ng) Example 2 Reference Example 2 1 0.0090 0.020010
0.0380 0.1360100
0.2920 0.17351.000
0.2g70 0.155010 00
0 0.2655 0.2325 From the above results, when poly A of 1100n or more is used, Example 2 in which it was pre-hybridized with the fixed oligo dTl9-14 made it easier to perform measurements without pre-hybridization. Higher sensitivity than in Reference Example 2 was obtained.

Example 3 Using a hybridized product prepared by hybridizing 1100n polyA to the solid-phase oligo dT 9-24 obtained in Example 2 and washing it according to the method of Example 1 (2), Example 2, 37°C118
A time reverse transcriptase reaction was performed.

At this time, the presence i1 of RAV-2-derived reverse transcriptase is O=
0.01.0.02.0.04.0.08, o, 16.
0.31, 0.63.1.25.2.5 and 5mU
(Above, each solution volume was 50 μl),
Thereafter, absorbance was measured according to Example 1 (4).

The results are shown in Table 4 below.

Reference Example 3 2-foil reaction buffer 50 μm described in Example 1 (3)
0.24 nmol of piotinylated-11-dUTP,
0.96 nmol dTTP and 1. Solid phased oligodTt was prepared in Example 2 by adding OOOng polyA. -24 wells each. P for this
RAV-2-derived reverse transcriptase was prepared in a solution volume of 50 μl in BS at 0,0.01.0.02.0.04.0.08.
0.16.0.31.0.63.1.25.2.5 and 5 mU were added, and a reverse transcriptase reaction was performed at each addition amount.

Then, the absorbance was measured according to Example 1 (4).

The measurement results of Example 3 and Reference Example 3 are shown in Table 4,
As shown in the table, in Example 3, reverse transcriptase was 0.0
While it can be measured from around 4 mU, in Reference Example 3 it is 0.0
The result was that it was difficult to measure even at 8 mU.

(Margin below) Table Q Q 00.01
0.015 0.0130.02
0.021 0.0230.04
0.0g2 0.0270.08 0
．． 159 0.0650.16 0.3
51 0.1220.31 0.752
0.3010.63 1°167
0.6381.25 2.239
1.8012.5 2.476 2.
1275 2.777 2.625
λ = 405 nm Example 4 Amount of immobilized oligo d Tl9-24 optimal for reverse transcriptase reaction and polyA hybridized thereto
In order to determine the most efficient V) primer to template ratio for reverse transcriptase reaction, the following experiment was conducted.

(1) Real oligo dT19 binding to aminoplate
-24 Determination of amount ■Labeling of oligo dT 19-2a 10 μg of oligo dTIo-2a was dissolved in 20 μm of water, and this was added with 10 times the concentration of caination buffer (5
00mM) Lis-HCl buffer (pH 7-, 6), 100
2 μl of mM MgCl2.100 mM 2-mercaptoethanol), 10 μCi/μl of [γ-32P]
Oligo dT 1G-2a was prepared by adding 1 μl of ATP, 20 units/μl T, and 1 μm of polynucleotide kinase, and incubating at 37°C for 30 minutes.
The 5° end of the protein was labeled with [γ-32P]. After 30 minutes, 2 ng of the reaction product was developed by paper chromatography (developing solution was 350 tpM ammonium formate),
After drying, the filter paper was divided into five equal parts, and it was confirmed that there was radioactivity at the origin, that is, oligo dT was labeled with [γ-32p]. Unfold and divide the filter paper into 5 equal parts 1.2 from the origin
．． They were numbered 3.4.5 and the measured radioactivity is shown in Table 5.

Table Developed Fraction Number Radioactivity 1 (Origin') 4587 91% of the radioactivity is at the origin, that is, in both regions where oligo dT1 and 24 are localized, and oligo dT.

It was confirmed that No. 24 was labeled with [γ-32P].

Also, from Table 5, labeling rate: 91% specific activity: 2.3×10′ cpm/μg was calculated.

Add another 30 μl of water to the remaining reaction solution to bring the total volume to 50 μl.
l, and add an equal amount of phenol (1mM EDTA in advance) to this.
10mM Tris-HCl buffer (pH 7.5) containing
After stirring, the aqueous layer was collected. To this was added 5 μl of 3M sodium acetate and 15 μl of 3M sodium acetate.
0 μl of cold ethanol was added and cooled at −80° C. for 15 minutes. After centrifugation, the supernatant was removed and the remaining pellet was washed twice with cold 75% ethanol.

■ Immobilization of oligo dT 19-2a on an amination plate and quantitative determination of bound oligo dTlCl-24 Vacuum dry the remaining pellet (labeled oligo dT) and add 0.1μ of water.
Dissolve to give a concentration of 2 g/μl, and add 2.
g/50ul, 200ng150μl, 20n
The solution was diluted with the aqueous solution containing CDI and IMD described in Example 1 (1) to give 150 µl of g and 2 ng/50 µl, and 50 µm of the solution was dispensed into amino plates divided into wells, and incubated at room temperature for 24 hours. Made it react. After the reaction, the wells were washed as described in Example 1 (1), and the radioactivity remaining in the wells was measured. When each amount of labeled oligo dT was used, the radioactivity remaining in the well of the aminobrate and the specific activity of labeled oligo dT (2,3 x lO'cpm/μ
g) and the actual amount (weight and number of moles) of oligo dT+o-2a bound to the aminobrate calculated from the average molecular weight (7,095 Daltons) are shown in Table 6, and the oligo dT19-24 reaction solution is contacted. Table 6 shows the amount of oligo dT+o-24 bound per unit area calculated from the surface area of the well (0.63 cm2).

(Margin below) Table 2 +, 637 0.71
0.10020 13.003
5.67 0.799200 2
6.555 11.58 1.6332
, OQO47,79820J 2.933
Based on the above results, oligo dT bound to one well of the aminobrate. -24 amount is added oligo dT. −
24, and when 000 ng of oligo dTlo-za mira was added, 20 ng/
1 unit was combined.

One well of the amino plate is cylindrical with a bottom area of 0.32 cm2, and contains oligo d TIGI.
Since the total liquid volume for the binding reaction of −24 is 50 μm,
The surface area on the well to which oligo dT'+o-2a can bind is 0.63 cm2, and oligo dT. Although the average molecular weight of -24 (average chain length: 21.5 bases) is 7.095 daltons, the bound oligo d per well unit surface area
The amount of T was up to about 4.655 pmol/am2.

(2) Quantification of poly A that hybridizes to solid phase oligo dT ■ [γ-32P] labeling of poly A Dissolve 50 g of poly A in 100 μl of water, and add to this as described in Example 4 (1) ■ Add 10 μl of the caination buffer at 10 times the concentration, add 10 μCi/μl of [γ-”P
PolyA was labeled with [γ-32P] by adding 1 μm of EATP, 20 units/μl of T, and 2 μl of polynucleotide kinase, and incubating at 37° C. for 30 minutes. Using a 30 minute rule, 25 g of the reaction product was added to Example 4 (
1) Developed by paper chromatography in the same manner as in ①, and after drying the developed filter paper, divide it into 6 equal parts of 1 cm each from the origin and confirm that there is radioactivity at the origin, that is, polyA is
P] was confirmed to be labeled. The filter paper was expanded and divided into six equal parts, numbered 1, 2, 3, 4, 5, 6 from the origin, and the measured radioactivity of each is shown in Table 7.

Table Developed Fraction Number (Origin) Radioactivity (cpm) 85.8% of the radioactivity is at the origin, ie oligo dT. -2
Oligo dTlG-2a is present in the localized fraction of [γ
-32P] labeling was confirmed. Further, from Table 7, it was calculated that: Label rate: 85.8% Specific activity: 1.14×10'cpm/μg.

The remaining reaction solution was poured according to the method described in Example 4 (1) ■.
Protein was removed and ethanol precipitated.

■Hybridization of [γ-32P] labeled poly A and immobilized oligo dT Example 4 (2) The pellet obtained by deproteinization and ethanol precipitation according to ■ (1!Identified poly A) was vacuum-dried and washed with water. 1ag
Dissolve 10 μg/μm in 750 μl
, 1ag150μm, 100ng/50p
1, Long 150 μl and 1 ng/50
It was diluted with high salt concentration TBS so that it was .mu.m. Oligo dT of each member found in Example 4 (1) ■.

Add 50 μl of labeled polyA diluted to each concentration to the wells of the plate bound to 24, hybridize at 4°C for 18 hours, and then wash the wells 5 times according to the method in Example 1 (2). The radioactivity remaining in the wells was measured. When each member's labeled poly A was hybridized to each member's oligo dT+o-2a immobilized plate, the radioactivity remaining in the well and the specific activity of the labeled poly A (1,14 x 10'
c pm/μg) and average molecular weight (86,700 daltons).
Table 8 shows the weights (weight and number of moles) of labeled polys that actually hybridized with 2a, and the weights of each oligo d T , 2. Table 9 shows the amount of polyA hybridization per unit area calculated from the surface area (0.63 cm2) of the wells of the fixed plate.

(Left below) (3) Examination of the optimal amount of template and immobilized primer in DNA chain elongation reaction using reverse transcriptase PolyA from each company and each immobilized oligo dT+o found in Example 4 (2) ■ -24 hybridized product, 0.
Reverse transcriptase was allowed to act according to the method of Example 2 using 1 mU of RAV-2-derived reverse transcriptase. Example 1 (4)
An optimal immobilized oligo dT that can be used to measure elongated biotinylated DNA using the method described above, with the highest sensitivity for reverse transcriptase reactions. The amount of hybridization between -24 and polyA was investigated.

Each polyA and immobilized oligo dT. The molar ratio of the hybridized product of the combination of -24 amounts and each molar ratio of 0.
Table 9 shows the absorbance obtained when 1 mU of RAV-2-derived reverse transcriptase was allowed to act at 37°C for 18 hours.

From the above results, the optimal amount of immobilized oligo dT19-24 and polyA to obtain the lowest reliable absorbance (approximately 0.1) is determined by the amount of immobilized oligo dT IG-2- (average chain length: 21.5 bases, average molecular weight 7,095 daltons) amount from 5.6 ng (1,2 pmo 1/am”) to 20
ng (5 pmol/cm”), preferably 11.
It was about 5 ng (2.6 pmol/cm').

Also, immobilized oligo dT in an amount within this range. Solid-phase oligo dT to obtain an absorbance of about 0.1 at -24 1. The amount of polyA (average chain length: 255 bases, average molecular weight 86,700 daltons) to be hybridized with -24 is 19 ng (0.3 pmo 17 am2)
The amount was 567 ng (10 pmol/cm2), preferably 82 ng (1.5 pmol/cm2).

From these facts, the most preferable immobilized oligo dT+o-2a:polyA hybridization amount for reverse transcriptase reaction is 18 ng/cm": 130 ng/Cm
2, and the molar ratio was approximately 1:0.6.

Example 5 Poly A and poly dA were used as templates reverse transcriptase reaction Oligo dT prepared according to the procedure of Example 1 (1).

-24 fixed plate was hybridized with 1100n polyA or polydA according to the method of Example 1 (2) and washed to prepare a hybridized product. Using this hybridized product, 1 mU 150. ul of RAV-2-derived reverse transcriptase at 37
The elongated piotinylated DNA was measured by the method described in Example 1 (4) after being allowed to react at °C for 18 hours. Poly A in the mold
Table 10 shows the absorbance obtained when reverse transcriptase was activated using poly-dA.

Table 1: Template absorbance* Poly A 1.53 Poly dA O,012 In this way, reverse transcriptase does not act on poly dA, and reverse transcriptase can be specifically measured by using poly A as a template. Ta.

Example 6 Quantification of antibody blocking reverse transcriptase activity Mo1t-4 cells (supplied by Dainippon Pharmaceutical Co., Ltd.) were infected with human immunodeficiency virus (Human Immunodeficiency Virus).
virus; infected with HIV),
These cells were added to RPMI-16 supplemented with 10% tallow serum.
40 medium (manufactured by Gibco), carbon dioxide concentration 5%, 37°
The cells were cultured at C for about 2 weeks. Culture supernatant at 30,000 rpm
Virus particle pellets (106 particles) were centrifuged at m for 1 hour.
and add this to 0.5% Triton X-100,0,
A mild solution consisting of 8mM NaCl, 0.5mM phenylmethylsulfonyl fluoride, 20% glycerin and 50mM Tris-HCl (pH 7°8). It was suspended in solution i to obtain an enzyme solution.

On the other hand, serum obtained from an HIV-infected person was diluted 20 times with 50 mM ethylenediamine-acetate buffer (pH 7.0), and 1 ml of it was passed through a QAE-Sephadex A-50 column (Gelpet 1 ml). fraction 4ml
was used as a partially purified tunnel transcriptase antibody.

First, 25 μl of the above enzyme solution and 25 μm of antibody stock solution, 1/10 and 1/100 dilution solution, or PBS were mixed and incubated at 4° C. for 30 minutes.

Oligo dT prepared according to the procedure of Example 1 (1),
- Using the hybridized product prepared by hybridizing 1100n/poly A according to the method described in Example 1 (2) on the □4 fixing plate, and adding it to the mixed solution according to the method described in Example 2. The remaining reverse transcriptase activity was measured. As a result, the absorbance shown in Table 11 was obtained, and the mine shaft transcriptase antibody present in the serum was quantified.

Table 11 Antibody dilution factor Absorbance * ×1 × 10 × 100 (no antibody) 0.0 2 1 0.180 0.5 6 8 1.174 * ^ = 405 nm Example 7 Reverse transcription using different oligo dT chain lengths Enzyme reaction chain lengths are 8.10.12.15.12 to 18.
Example 1 (200 ng each of oligo dT 19-24, 25-3o and 272 (all manufactured by Pharmacia)
It was immobilized on an amino plate by the method described in 1), hybridized with each immobilized oligo dT using 100 nH polyA according to the method described in Example 1 (2), and washed to obtain each hybridized product. was prepared. Each hybridized product was treated with 0.1 mU of reverse transcriptase under the conditions of Example 2, and the elongated biotinylated DNA was measured according to the method described in Example 1 (4). Table 12 shows the relationship between the length of oligo dT and the degree of absorbance. oligo d
The absorbance increased depending on the length of the T chain. The length of the chain is 10
The results below are not preferred, but the results are preferred when the chain length is 12 or more. Therefore, it was confirmed that chain lengths of 11 or more are usually used.

No. table oligo dT absorbance book 12-18 19-24 25-30 0.0 2 9 0.0.62 0.118 0.146 0.152 0.2 9 1 0.501 0.6 2 1 Example 8 Biotinylated DN depending on the amount of dTTP added Change in detection sensitivity of A Oligo dT fixed as described in Example 1 (1).

-24 fixed plate by the method of Example 1 (2).
oong poly A was used for hybridization, and this was mixed with the reaction buffer 4 of 2 times the strength described in Example 1 (3).
6 μl was taken out. On the other hand, biotinylated-11-dUTP
and dTTP at a mixing molar ratio of 1:0.1:3, respectively.
．． 1;4.1:5゜7.1:9.1: 12.3.1
:19, 1:24.1:29.1:39.1:49 and 1:99, and these mixtures were combined with biotinylated-11-dUTP and dTTP to a final concentration of 0. The concentration was adjusted to 3mM. These 0.3mM
A 4 μm thick mixture of biotinylated-11-dUTP and dTTP was added to the oligo dT'+o-2a immobilized plate hybridized with polyA, and mixed with the reaction buffer. Furthermore, 50 μl of RAV-2-derived reverse transcriptase prepared at 1 mU 150 μm in PBS was added to each well, mixed, incubated at 37° C. for 18 cycles, and biotinylated according to the description in Example 1 (4). D.N.
A was quantified. Piotinylated-11-dUTP and dTTP
Table 13 shows the absorbance at each mixing molar ratio.

Biotinylated dUTP: dTTP absorbance 1 mixed molar ratio As is clear from this result, biotinylated-11-dU
The best sensitivity was obtained when the molar ratio of TP and dTTP was 1:4.

Below Up Out wish Man Toyo Jozo Co., Ltd.

Claims (10)

[Claims] (1) At least a hybrid in which an artificially prepared adenine ribopolynucleotide template RNA and an immobilized oligodeoxythymine nucleotide complementary to the adenine ribopolynucleotide template RNA are hybridized in an aqueous medium. The soybean product and biotinylated deoxyuridine triphosphate are reacted in the presence of a test solution, and the reaction product of the reaction and unreacted biotinylated deoxyuridine triphosphate are separated, and then the reaction product or unreacted biotin is separated. 1. A method for determining reverse transcriptase in a test liquid, the method comprising measuring the amount of deoxyuridine triphosphate. (2) The solid phased oligodeoxythymine nucleotide is a solid phased oligodeoxythymine nucleotide in which the 5' end of the oligodeoxythymine nucleotide and the solid phase are chemically bonded. How to decide. (3) The determination method according to claim 1, wherein the immobilized oligodeoxythymine nucleotide consists of nucleotide residues of 11 bases or more. (4) Immobilized oligodeoxythymine nucleotide is 1.2 pmol/cm^2 to 5.0 pmol/cm
^2, adenine ribopolynucleotide template RNA
The determination method according to claim 1, wherein is hybridized at a molar ratio of 3 to 0.07, where 1 is immobilized oligodeoxythymine nucleotide. (5) The determination method according to claim 1, further comprising the step of containing deoxythymidine triphosphate during the reaction. (6) At least a hybrid in which an artificially prepared adenine ribopolynucleotide template RNA and an immobilized oligodeoxythymine nucleotide complementary to the adenine ribopolynucleotide template RNA are hybridized in an aqueous medium. The soybean product and biotinylated deoxyuridine triphosphate are reacted in the presence of a test body fluid and reverse transcriptase, and the reaction product of the reaction and unreacted biotinylated deoxyuridine triphosphate are separated, and then the reaction product or A method for confirming retrovirus infection in a subject, comprising measuring the amount of unreacted biotinylated deoxyuridine triphosphate and determining the reverse transcriptase neutralizing antibody titer in the subject's body fluid from this amount. (7) The solid phased oligodeoxythymine nucleotide is a solid phased oligodeoxythymine nucleotide in which the 5' end of the oligodeoxythymine nucleotide and the solid phase are chemically bonded. Infection confirmation method. (8) The method for confirming infection according to claim 6, wherein the immobilized oligodeoxythymine nucleotide consists of nucleotide residues of 11 bases or more. (9) Immobilized oligodeoxythymine nucleotide is 1.2 pmol/cm^2 to 5.0 pmol/cm
^2, adenine ribopolynucleotide template RNA
7. The method for confirming infection according to claim 6, wherein hybridization is carried out by mixing and hybridizing at a molar ratio of 3 to 0.07, with the immobilized oligodeoxythymine nucleotide being 1. (10) The method for confirming infection according to claim 6, further comprising the step of containing deoxythymidine triphosphate during the reaction.