JP2715851B2 - Nucleic acid detection method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a D
A method for detecting NA or RNA, which is used for diagnosing a disease or genetic disease caused by a virus or a bacterium, or for identifying a species or discriminating between a parent and a child.

[0002]

2. Description of the Related Art Hybridization is effective for diagnosing diseases caused by gene mutation and diseases caused by viruses.

[0003] Conventionally, detection has been carried out by using physical methods such as ultrasonic crushing of target DNA or RNA from bacteria, viruses, leukocytes, etc., using enzymes such as proteinase K, and surfactants such as SDS. Extract by chemical method. After that, it was treated with phenol and chloroform, and precipitated with ethanol.
After purifying A (RNA), it is denatured with an alkali such as NaOH to obtain single-stranded DNA (RNA). A well-known filter hybridization method is to fix the single-stranded DNA (RNA) for this purpose on a membrane filter (nitrocellulose, nylon).

When an oligonucleotide probe labeled with a radioisotope, an enzyme, biotin, a fluorescent substance, or the like is added to a filter on which the DNA (RNA) is immobilized, if the oligonucleotide has a sequence complementary to the target DNA (RNA), A hydrogen bond occurs between the two to form a double strand. Oligonucleotide probes that have no or no complementary strand are removed from the filter by washing. After that, the objective D was determined by measuring the radioactivity, enzyme activity,
Detection of NA (RNA) is performed.

[0005] In recent years, polymerase chain reaction (PCR)
(Saiki, RK et al, Science 239 487-491 (1988)). This method can amplify only a target DNA region from a very small amount of a gene (DNA) by about one million times within several hours. That is, when DNA primers (18 to 30 nucleotides) for the + and − strands are synthesized across the gene region to be amplified and the synthesis of DNA fragments is repeated by the DNA polymerase, the DNA is amplified twice in each cycle. After n cycles, 2
Amplified ^n- fold. At this time, by selecting a sequence specific to the target gene as a DNA primer, a highly selective polymerase reaction occurs and the primer
A single-stranded DNA fragment is produced. In this method for detecting a double-stranded fragment, it is a general method to bring DNA amplified by a PCR reaction (amplified DNA) into the above-described filter hybridization method.

[0006] As another method, an affinity matrix in which a primer is labeled with biotin in advance, a PCR reaction is performed, a DNA probe labeled with a radioactive substance is hybridized in a liquid phase, and then avidin is immobilized. Detection method using (Ann-Chr
istine Syvanen et al, Nucl. Acids Res. 16 (1988) 113
27-38), a method in which PCR is performed, hybridization is performed with two types of DNA probes, and then adsorption is performed on a solid phase for detection (Patrik, O. Dahlen et al, J. clin. Microbiol.
(1991) 798-804), or Adler et al.
No. 99 etc.

[0007] A method of directly analyzing amplified DNA by electrophoresis is also known. In this method, a DNA fragment labeled or not labeled with a radioisotope is electrophoresed in an acrylamide gel carrier, and the gel is subjected to autoradiography, or the gel is placed on a thin-layer silica gel plate, and the gel is placed in a gel with a UV lamp. And take a picture. Alternatively, a DNA fragment is electrophoresed in an agarose gel carrier, the DNA is stained with ethidium bromide, the gel is placed on a transilluminator (which emits ultraviolet light) from the electrophoresis tank, and a photograph is taken (T. Man
iatis et al, Molecular Cloning, Cold Spring Harbo
ur (1982)).

[0008]

However, in the method using a filter, a DNA (RNA) is used for the filter.
, An operation of fixing DNA (RNA) by heat or ultraviolet rays, and a complicated operation of washing an oligonucleotide probe that did not form a complementary strand. In addition, there is another inconvenience that the DNA probe is non-specifically adsorbed on the surface of the filter, thereby lowering the detection sensitivity and increasing the background.

[0009] In addition, after carrying out a PCR reaction by labeling each primer with biotin in advance, and performing hybridization in a liquid phase with a DNA probe labeled with a radioactive substance, an affinity matrix on which avidin is immobilized is prepared. In the method of detection using the method, the operation is complicated since the hybridization is carried out and then immobilized on a carrier, and the conditions for ordinary PCR reactions need to be changed in order to label the primers. Furthermore, the use of a large amount of label primers during PCR is costly.

In the above method, the primer 2
A total of four specific sequences, two species and two DNA probes, were required, and the selection and synthesis of the sequences became very complicated.

[0011] Furthermore, the reaction process is multi-step, and centrifugation is required.

In the electrophoresis method, when a radioactive substance is used, a special facility is required, and the gel cannot be stored for a long period of time. Also, it takes one and a half hours or more after the end of PCR.

Accordingly, the present invention provides a method for detecting nucleic acids (DNA, RNA) which overcomes these problems, enables high-sensitivity multi-sample processing in a short time, and produces an objective output result without any trouble. The purpose is to do.

[0014]

Means for Solving the Problems In order to solve the above-mentioned problems, the present invention allows at least one kind of oligonucleotide to function as a chain length reaction primer to selectively use a DNA synthase in a specific DNA in a target DNA. (First reaction), a reagent or a temperature for inactivating DNA synthase, and a labeled primer having one sequence of the primers used for the generation are allowed to act to denature the generated gene Simultaneously, a step of attaching a labeled primer to the produced gene (second reaction), and reacting the reaction product produced in the second reaction with the labeled oligonucleotide having a sequence complementary to a partial sequence of the produced gene and the second reaction Simultaneously reacting with the solid phase recognizing the labeled primer (third reaction), the signal generated from the labeled oligonucleotide used in the third reaction It consists a step of detecting a presence of the target nucleic acids by performing constant (fourth reaction).

Here, the first reaction is performed by a gene amplification method, for example, a PCR reaction (Saiki et al., Science (230) 1350-4), LCR
(Barany et al., Proc, Natl. Acad. Sci. USA (88) (89-93)),
Alternatively, a known method such as a primer extension method using a DNA enzyme is used. When a PCR reaction is used, a method using a larger amount of one kind of primer than the other (Asymmetric PCR) is preferable. Examples of the primer used in the PCR reaction include, but are not limited to, those of Japanese Patent Application No. 3-59820 by the present applicant. Combinations of two oligonucleotides, for example, when detecting Vibrio parahaemolyticus thermostable direct hemolysin genetic <br/> child a (tdh gene), (a) 5'dGGTACTAAATGGCTGACAT
C (SEQ ID NO: 1) and (b) 5'dCCACTACCACTCTCATATG
When detecting the cholera toxin gene using C (SEQ ID NO: 2), (e) 5 ′ dACAGAGTGAGGTACTTTGAC
C (SEQ ID NO: 5) and (f) 5'dATACCATCCATATATTTGGG
GAG (SEQ ID NO: 6) is used.

The reagent for inactivating the DNA synthase used in the second reaction includes, for example, EDTA, and the label of the labeled primer includes biotin. Not limited.

In order to denature the amplified gene, the temperature of the amplified gene solution is raised to 90 ° C. to 95 ° C. or an alkaline solution such as NaOH is used.

The nucleotide sequence of the labeled oligonucleotide used in the third reaction, for example, when detecting Vibrio parahaemolyticus thermostable direct hemolysin <br/> gene (tdh gene) when using (b) the second reaction Is (c) 5'dCCAAGTAAAAGTGTATTTGG
A (SEQ ID NO: 3) was combined with (a)
When using (d) 5'dTCCAAATACATTTTACTTG
G (SEQ ID NO: 4) can be used. When the cholera toxin gene is detected, (f) is used in the second reaction when (f) is used in the second reaction. (H) 5'dTTCCACCCTCAATTAGTTTG
When (e) is used in the second reaction as the sequence that binds to AGAA (SEQ ID NO: 7), a sequence that binds to the complementary strand of (h) can be used.

Further, an enzyme or a fluorescent dye can be used as the label of the labeled oligonucleotide, and examples of the enzyme include, but are not limited to, alkaline phosphatase and peroxidase. As the fluorescent dye, for example, FITC,
Rhodamine can be mentioned.

As a solid phase for recognizing the labeled primer, for example, a 96-well microplate to which streptavidin or avidin is fixed is used.

The measurement in the fourth reaction is performed using an enzyme substrate when the label of the labeled oligonucleotide in the third reaction is an enzyme. The enzyme substrate is, for example, 4-methylumbelliferyl phosphate or p-nitrophenyl phosphate when the enzyme is alkaline phosphatase, or parahydroxyphenylpropionic acid or 3,3 ′, 5,5′- when peroxidase is used. Tetramethylbenzidine and O-phenylenediamine are used.

When the label is a fluorescent dye, the fluorescence generated by irradiating a laser beam is measured. When the target nucleic acid is RNA, the reaction is carried out by transferring the DNA to DNA using reverse transcriptase.

[0023]

According to the present invention, the presence or absence of a target nucleic acid can be determined with high sensitivity without electrophoresis and in a short time without using a radioisotope. In addition, when a tool such as a multi-well microplate that can process a large amount of sample at a time is used, it becomes possible to automate the detection of a target nucleic acid.

[0024]

【Example】

(Example 1) Vibrio parahemolyticus which is the first reaction Kanagawa phenomenon positive strain
The WP1 strain was cultured in liquid and total DNA was extracted by a conventional method using phenol and chloroform. The amount of the DNA was quantified from the value at 260 nm using an ultraviolet-visible absorption spectrophotometer. Serially dilute this solution 4fg, 40fg, 400fg, 4000fg / 3ul
TE solution (10 mM Tris-HCl buffer containing 1 mM EDTA, pH
8.0) and used for PCR. Chromosomal DNA 4fg
Was used in the following experiment as one copy.

The PCR reaction conditions were 10 × buffer 3 μm.
l, dNTP 4.8 μl (1.25 mM each), primer having a sequence specific to the Vibrio parahaemolyticus tdh gene (a) 0.75 μl (25 nmol / ml), (b) 0.75 μl (2.5 nmol / m
l) Nonidet P-40, Tween 20, 0.5% each 3ul, and heat resistant DNA polymerase 0.15ul (5unit / ul) were added to prepare 30ul of reaction solution.

To the container containing the reaction solution, 50 μl of mineral oil (SIGMA) was added to prepare a reaction solution. The composition of each buffer is shown below.

10x buffer: 500mM KCl 100mM Tr
is HC1 (pH 9.0) 15 mM MgCl ₂ , 0.1% gelatin, dNTP solution: dATP dCTP dGTP dTT
Refers to a mixture of P.

The reaction conditions are as shown below.

Thermal denaturation: 94 ° C., 1 minute Annealing: 55 ° C., 1 minute Polymerization reaction: 72 ° C., 1 minute, 35 cycles were performed.

The sequence of each primer is a sequence reported to the 64th Annual Meeting of the Bacteriological Society of Japan (Tada et al., JJBacteriol 199146 281). Sequence is primer (a) 5'dGGTACTAAATGGCTGACA
TC3 '(b) 5' dCCACTACCACTCTCATATGC3 'and Vibrio
M. Nishibuchi et al. (1990) Mol. Micro is a primer that can specifically detect the parahemolyticus tdh gene.
The sequence in the tdh2 gene described in biology 4 87-99 is used. Both primers (a) and (b) were prepared using a DNA synthesizer manufactured by Milligen, and purified by reversed-phase high performance liquid chromatography and used for PCR.

A labeled primer in which biotin was labeled at the 5 'end of the sequence of the second reaction primer (b) was prepared. This is the primer (b)
Was reacted with a monomethoxytritylamine linker to bind hexanolamine (amino group) to the 5 'end, and then oligonucleotide (25 nmol, 100 ul) 15 mM N-hydroxysuccinimidobiotin (1 nm) dissolved in 1 × PBS (PH7.4) (DMF solution) by reacting with 150 ul. Purification was performed by a gel filtration method using a PD-10 column (Pharmacia).

50 mM E containing 3.8 pmol of a labeled primer labeled with biotin at the 5 'end of the sequence of the primer (b)
30 μl of the DTA solution was added to each tube containing 30 μl of the solution after the PCR reaction, heated at 95 ° C. for 5 minutes, and then heated to 25 ° C.

The operations of the first and second reactions were performed in a thermal cycler for PCR and performed in the same single tube.

Third reaction 96-well microplate on which streptavidin is immobilized
(C) 5'dCCAAATCACTTTTACTTGG3'100 pmol / oligonucleotide (c)
2 x SSC (1 x SSC is 0.15M, NaC
l, 0.015 M citrate-3-sodium), previously put 1% Tween solution 30ul of distilled water 75 mu l, T E solution 75 mu l solution was added, samples were added 4 mu l of the second reaction After holding at room temperature for 1 hour, the microplate was washed.

The fact that the sequence of the oligonucleotide (c) used here is specific to the tdh gene was confirmed by J, Tada et al., Mol. Cel.
l.Probe.1992.5477.

The method for preparing the peroxidase-labeled oligonucleotide used in the reaction is described in Murakami, A. et al, Nuc.
l.Acid Res. 1989.17.5587-5595).
That is, an oligonucleotide having a thiolated 5′-position
(c) SPDP-peroxidase (17 nmol) obtained by reacting 22 nmol with horseradish peroxidase (Boehringer) with SPDP was finally added to 1 × PBS (pH 7.4) 200
ul and reacted at room temperature overnight. Thereafter, a Biogel p-60 (BioRad) column (1.0 swelled with 0.5 × PBS) was used.
x45 cm) and eluted with 0.5 × PBS. Thereafter, the absorbance at 403 nm and 260 nm was measured, and fractions having a molar ratio of DNA to peroxidase of 1: 1 were collected. At this time, ε403 (unit: l · cm ⁻¹ · mol ⁻¹ ) of peroxidase = 91,0
00, ε260 = 28,000, ε260 of DNA = 200,000 ε403
= 0 (ε is molar spectral extinction coefficient). Final concentration after fractionation
It was made into a 1 × PBS solution containing 0.01% BSA (bovine serum albumin, Sigma fraction V) and stored at 4 ° C. until use.

A 96-well microplate immobilized with streptavidin was prepared as follows. That is, bovine serum albumin (BSA, Fraction V, Sigma) 700n
The mol was dissolved in 500 ul of 1XPBS, and 4.6 mg of biotinamide caprosan (BRL, dimethylamide solution) was added, followed by reaction at room temperature for 2-3 hours.

The reaction solution was purified by a G-25 gel filtration column (PD-10, Pharmacia). The solution was dissolved in 1XPBS solution and dissolved so that the absorbance at 280 nm became 1.0 OD / ml.
200ul was added to the well microplate. After leaving at 4 ° C. overnight, the solution was discarded and 200 μl of 1% BSA was added to each well to perform blocking at 4 ° C. overnight. The solution was discarded, and the wells were washed twice with 1XPBS containing 0.1% Tween 20. Then, 20 ug / ml of streptavidin (BRL) was added to each well and allowed to stand at room temperature for 2-3 hours. Thereafter, the wells were stored at 4 ° C. until use, and the wells were prepared by washing the wells twice with 1XPBS containing 0.5% Tween 20 immediately before use.

The solution in the well is discarded and the plate is then washed with 200 ul of 1 × PBS containing 0.5% Tween 20 at room temperature.
The plate was washed twice for 1 minute, once for 1 minute with 200 ul of 1 × PBS containing 0.5% Tween 20 previously heated to 40 ° C., and once again for 1 minute at room temperature.

Fourth reaction 0.1 containing 0.56 mM 3,3 ', 5,5'-tetramethylbenzidine
The reaction was stopped by adding 150 ul of M acetate buffer (PH5.5) and 50 ul of 0.02% hydrogen peroxide solution, keeping the mixture at room temperature for 30 minutes, and adding 25 ul of 1 M sulfuric acid. The absorbance of the solution in the microplate was measured at 450 nm using an immunoreader NJ-2000 (Intermed).

As a result, as shown in FIG. 1, 10 copies of Vibrio parahaemolyticus WP-1 strain tdh gene could be detected.

Example 2 In order to examine the sensitivity of this method, a comparison was made with a conventional agarose electrophoresis method. First,
The reaction conditions were 35 cycles of 94 ° C. for 10 seconds, 55 ° C. for 10 seconds, and 72 ° C. for 15 seconds. Other conditions were performed in the same manner as in Example 1, and then 5 ′ position of the primer having the same sequence as one of the primers used in the PCR reaction.
2.6 pmol of primer labeled with biotin in the same manner as
20 μl of a 50 mM EDTA solution containing PCR (after removing the solution used for agarose electrophoresis)
20ul), heated at 95 ° C for 2 minutes and cooled to 25 ° C.

4 μl of the tube was previously immobilized with streptavidin and added to a 96-well microplate containing the following solution.

Solution contained in 96-well microplate: 20
Immobilization of DNA on a 96-well microplate having a total volume of 200 ul containing 0.3% SSC, 0.15% Tween 20 containing pmol / ml of peroxidase-labeled oligonucleotide, and hybridization for 60 minutes, washing 1 (1x PBS, O.D. 5% Tween 20
200 ul) for 1 minute, washing 2 (1xPBS, O.5% Tween 20
(Solution heated to 40 ° C.)) for 1 minute, washing 3 (1 × PBS, O.
5% Tween 20 200ul) for 1 minute, then 0.56mM 3,3 ',
0.1 M acetate buffer containing 5,5'-tetramethylbenzidine (p
H5.5) 150ul, and 50ul of 0.02% hydrogen peroxide were added and the enzymatic reaction was performed at room temperature for 30 minutes.

After the reaction, 25 ul of 0.2N sulfuric acid was added to stop the reaction, and the absorbance at 450 nm was measured with an immunoreader. The result is shown in FIG. FIG. 2 (a) shows that according to the present invention, about 10 copies of the tdh gene of Vibrio parahaemolyticus WP-1 strain can be detected in a short time of about 1.5 hours of PCR reaction time and 1.5 hours after that (DNA detection time). (No. 3 in FIG. 2A).

FIG. 2 (b) shows the results of detection of the DNA after PCR by agarose electrophoresis for comparison. In addition,
The numbers in FIG. 2B and the numbers in FIG. 2A that are the same are the results of detecting the same PCR-amplified DNA by each method. In FIG. 2B, M is the φX174Hinc II DNA marker, and the position of the arrow is the position of the DNA amplified by PCR (250
Base pairs).

From these figures, it can be seen that the method of the present invention is one to two orders of magnitude more sensitive than agarose electrophoresis.

(Example 3) Added after the PCR reaction (second
The experiment was performed by changing the amount of the oligonucleotide (primer) added in the reaction. The PCR reaction used 100 copies of Vibrio parahaemolyticus WP strain DNA.

The procedure was the same as in Example 1 except for the amount of the oligonucleotide (primer) added after the PCR reaction. The amount used was 15 ul of 50 mM EDTA solution, sample 1; 1.9 pmol sample 2, 1.0 pmol, Sample 3;
5 pmol of biotin primer was used. In addition, 15 ul of the solution after the PCR reaction was used.

As shown in FIG. 3A, it was found that when the amount of the oligonucleotide (primer) was reduced, the signal per 1 ul of the reaction solution was reduced.

However, the volume of the solution used for the microtiter plate assay was set to 1; 3 ul, sample 2;
The signal did not decrease even when the amount of the oligonucleotide was decreased by increasing the amount to 6 ul, sample 3; 12 ul (FIG. 3 (b)).

Thus, according to the present invention, even if the amount of the labeled primer used is extremely small (about 1/20 of the amount used in a normal PCR reaction), the target DNA can be obtained without impairing the sensitivity. Can be detected.

Example 4 The number of strains used in the experiment was increased to examine the specificity of the present method (43 strains shown in Table 1 were used in the experiment).

The sample used for PCR was L.
Take 10 μl of the bacterial cell fluid cultured with shaking in B medium, add 90 μl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), and add
After heating for 5 minutes and centrifuging at 5000 rpm for 1 minute, 3 ul of the supernatant was used to label the primer in the second reaction with 0.1 pmol of a 50 mM EDTA solution.
Mix 20 ul and 20 ul of the solution after the PCR reaction and use 30 ul of them,
In the third reaction, the DNA was fixed and hybridized to the microplate at room temperature for 15 minutes, and the enzymatic reaction was performed for 30 minutes.
The procedure was performed in the same manner as in Example 1 except that the procedure was performed at 15 ° C. for 15 minutes.

Experimental Results As shown in Table 1, the target td
Large measurements were obtained only in the strains carrying the h gene (1.179 to 1.908), and other Vibrio spp .
micus, V. furnissii , V. cholerae ) obtained small values (-0.006 to 0.023). This indicates that this method can specifically detect the tdh gene.

[0056]

[Table 1] (Example 5) The cholera toxin gene was detected by this method. The strains used were: V. cholerae O1 eltor Ogawa ctx (cholera toxin gene) + (9 strains), V. cholerae O1 eltor Inaba ctx + (7 strains), V. cholerae O1 classical Inaba c
tx + (3 shares), V.cholerae O1 classical Ogawa ctx +
(2 shares), V.chol erae O1 eltor Ogawa ctx- (2
Co., Ltd.), V.cholerae O1 eltor Inaba ctx - (3 share), V.
cholerae non O1 ctx + (143 strains), V.choleraen
on O1 ctx-(16 shares), E.coli (LT +) (6 shares)
There are 91 shares.

In this experiment, 5 ′ dACAGAGTGAGGTACTTT was used as a primer for the first PCR reaction.
GACC (SEQ ID NO: 5) and 5'dATACCATCC
Using the sequence of ATATATTTGGGAC (SEQ ID NO: 6), using the primer of SEQ ID NO: 6 as the labeling primer for the second reaction, and 5′dTTCCACCTCAATTTAGTTTG as the labeling oligonucleotide for the third reaction
The experiment was performed in the same manner as in Example 1 except that a sequence complementary to AGAA (SEQ ID NO: 7) was used (however,
DNA immobilization / hybridization on a microplate is the same as in Example 3). In addition, the above sequence is Mekalano
s, JJ et al. The sequence of Nature (1983.306.550) was used.

FIG. 4 shows the experimental results. As can be seen from the figure, the cholera toxin gene-positive strain has an absorbance of 0.55 to 1.73.
It can be seen that the value is 0.000 to 0.042 for the negative strain. Thus, the results show that the method is specific for the cholera toxin gene. The above explanation is based on the asymmetric P
As for CR, ordinary PCR can be performed by equimolar addition of a pair of primers in the first reaction, and the same result as described above can be obtained at this time.

[0059]

According to the present invention, since no labeled primer is used during PCR, existing primers can be used without changing the conditions, and hybridization of the labeled oligonucleotide and solid-phase immobilization are performed simultaneously.
The reaction time and operation steps up to detection can be reduced.

[0060]

[Sequence list]

Sequence number (SEQ ID NO); 1 Sequence length 20 bases Sequence type Number of nucleic acid strands Single strand Topology Linear Sequence type Genomic DNA Hypothetical sequence NO Antisense NO Origin of Vibrio parahaemolyticus Sequence Features The determined method S sequence GGTACTAAATGGCTGA
CATC

Sequence number (SEQ ID NO); 2 Sequence length 20 bases Sequence type Number of nucleic acid strands Single strand Topology Linear Sequence type Genomic DNA Hypothetical sequence NO Antisense NO Origin Vibrio parahaemolyticus sequence Features Method for determining features S Sequence CCACTACCACTCTCAT
ATGC

SEQ ID NO: 3 Sequence length 20 bases Sequence type Number of nucleic acid strands Single strand Topology Linear Sequence type Genomic DNA Hypothetical sequence NO Antisense NO Origin of Vibrio parahaemolyticus sequence Features Method for determining features S Sequence CCAAGTAAAATGTATT
TGGA

SEQ ID NO: 4 Sequence length 20 bases Sequence type Number of nucleic acid strands Single strand Topology Linear Sequence type Genomic DNA Hypothetical sequence NO Antisense NO Origin Vibrio parahaemolyticus sequence Features Method of determining features S Sequence TCCAAATACATTTTTAC
TTGG

SEQ ID NO: 5 Sequence length 20 bases Sequence type Number of nucleic acid strands Single strand Topology Linear Sequence type Genomic DNA Hypothetical sequence NO Antisense NO Origin V.cholerae sequence The method of determining the characteristic S sequence ACAGATGGAGTACTTT
GACC

SEQ ID NO: 6 Sequence length 22 bases Sequence type Number of nucleic acid strands Single strand Topology Linear Sequence type Genomic DNA Hypothetical sequence NO Antisense NO Origin V.cholerae sequence Characteristic method Method for determining characteristics S sequence ATACCATCCATATAT
TGGGAG

SEQ ID NO: 7 Sequence length 23 bases Sequence type Number of nucleic acid strands Single strand Topology Linear Sequence type Genomic DNA Hypothetical sequence NO Antisense NO Origin V.cholerae sequence The method of determining the characteristic S sequence TTCCACCTCAATTAGT
TTGAGAA

[Brief description of the drawings]

FIG. 1 is a diagram showing the detection of the DNA of the Vibrio parahaemolyticus WP-1 tdh gene according to the present invention.

FIG. 2A is a diagram in which DNA after PCR is detected by the present invention, and FIG. 2B is a diagram in which DNA after PCR is detected by agarose electrophoresis.

FIG. 3 (a) is a diagram showing an experiment in which the amount of oligonucleotide (primer) added after the PCR reaction (added in the second reaction) was changed, and FIG. 3 (b) was obtained by increasing the amount of solution used in the microtiter plate assay. Figure detected

FIG. 4 is a diagram in which DNA of the cholera toxin gene is detected according to the present invention.

Claims (4)

(57) [Claims] 1. A step (first reaction) of causing at least one kind of oligonucleotide to function as a chain length reaction primer to selectively generate a specific DNA in a target DNA using a DNA synthase (first reaction). A step of applying a labeled primer having a sequence of one of the primers used in the generation and the reagent or temperature to be activated to denature the generated gene and simultaneously attaching the labeled primer to the generated gene (second reaction) ), Simultaneously reacting the reaction product generated in the second reaction with a labeled oligonucleotide having a sequence complementary to a part of the sequence of the gene to be generated and a solid phase that recognizes the labeled primer acted on in the second reaction ( Third reaction), a step of detecting the presence or absence of the target nucleic acid by measuring a signal generated from the labeled oligonucleotide used in the third reaction (second reaction). 4 reactions). 2. The oligonucleotide according to claim 1, wherein the sequence of the oligonucleotide at the time of elongation in the first reaction is (a) 5 ′ dGGGTACTAAATGGCTGACAT.
C and (b) 5'dCCACTACCACTCTCATATG
C or one of the above, when both (a) and (b) are used, the labeled primer in the second reaction has the same sequence as nucleotide (b),
In the reaction, the compound binds to a labeled nucleotide having the following base sequence (c), and (c) 5 ′ dCCAAGTAAAATGTATTTGGG
A When either one of the above (a) and (b) is used in the first reaction, the second reaction has the same sequence as the nucleotide in the first reaction, and when (b) is used in the first reaction, , In the third reaction, binding to the above (c), and when (a) is used in the first reaction, binding to the sequence complementary to the above (c) in the third reaction. Item 4. A method for detecting the Vibrio parahaemolyticus thermotolerant hemotoxin gene ( tdh gene) according to Item 1. 3. The labeled primer used in the second reaction of claim 1 has the same sequence as nucleotide (a) of claim 2,
The nucleotide sequence of the labeled oligonucleotide in the third reaction is nucleotide (d) 5 ′ dTCCAATAATACATTTTACTTG
2. The method for detecting a Vibrio parahaemolyticus thermotolerant hemotoxin gene ( tdh gene) according to claim 1, comprising binding to G. 4. The oligonucleotide according to claim 1, wherein the sequence of the oligonucleotide at the time of elongation during the first reaction is (e) 5 ′ dACAGAGTGAGGTACTTTGAC.
C and (f) 5'dATACCATCCATATATTTGG
When GAG or one of the above is used, when both (e) and (f) are used, the labeled primer in the second reaction has the same sequence as nucleotide (f),
In the reaction, the compound binds to a labeled nucleotide having the following base sequence (h), and (h) 5 ′ dTTCCACCTCAATTAGTTTG
AGAA When (e) is used in the second reaction, it binds to the complementary strand of the above (h) in the third reaction, and when either of the above (e) or (f) is used in the first reaction, the second In the reaction, it has the same sequence as the nucleotide at the time of the first reaction, and when (e) is used in the first reaction, it binds to the sequence complementary to the above (h) and (f) in the first reaction 2. The method for detecting a cholera toxin gene according to claim 1, comprising binding to the sequence of (h) when is used.