JP3146565B2 - 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 DNA having a specific nucleotide sequence.
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 parent and 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. Thereafter, the objective D is determined by measuring the radioactivity, enzyme activity, and fluorescence intensity
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.

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-Chris
tine Syvanen et al, Nucl. Acids Res. 16 11327-11338
(1988)), oligonucleotide probes were labeled with a plurality of biotins, hybridized with the target RNA in a liquid phase, and then detected by adding an affinity matrix immobilized with avidin and an enzyme-labeled anti-RNA-DNA antibody. Method (Clifford O. Yehle et al, Molecular and Cell
uar Probes 1,177-193 (1987)).

[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. Mania
tis et al, Molecular Cloning, Cold Spring Harbor
(1982)).

A DNA sequencer is known as a method for determining the sequence of DNA (Smith, L, M, et al, N
ature 321 674-679 (1986)). This method uses fluorescently labeled D
DNA to be sequenced by fluorescent labeling of NA primer
In this method, the A-sequences are connected, electrophoresed on an acrylamide gel, and the fluorescence intensity derived from DNA is measured using an argon laser.

[0009]

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.

[0010] The DNA sequencer method can be used to amplify DNA.
When applied directly to the detection method, the DNA primer with a fluorescent dye that does not participate in the PCR reaction that causes a decrease in the detection sensitivity will enter the electrophoresis gel. In a DNA sequencer, it is sufficient to use a laser light source that works to increase the sensitivity High sensitivity cannot be earned. In addition, in this method, a large number of samples cannot be processed because data is taken in during electrophoresis.

[0011] In addition, after carrying out PCR reaction by labeling biotin to each primer in advance, hybridization is performed in a liquid phase with a DNA probe labeled with a radioactive substance, and then detection is carried out using an affinity matrix on which avidin is immobilized. (Ann-Christine Syvanen et.
al, Nucl. Acids Res. 16 11327-11338 (1988)), the operation is complicated because it is fixed to a carrier after hybridization. High sensitivity cannot be obtained unless a radioactive substance is used.

The use of radioactive materials requires special facilities and is expensive. They also require more than one and a half hours after the end of PCR. In addition, centrifugation is required for detection. They also use the same method using a fluorescent dye instead of a radioactive substance, but the sensitivity is inferior to the conventional method (for example, electrophoresis), and the desired sensitivity has not been obtained.

Therefore, the present invention overcomes these problems, enables high-sensitivity multi-sample processing in a short time without using a radioisotope, and provides a nucleic acid (DNA, DNA, RNA).

[0014]

Means for Solving the Problems In order to solve the above-mentioned problems, the present invention provides a method in which at least one of two types of oligonucleotides functions as a chain length reaction primer to convert a specific DNA in a target DNA into a DNA synthase. (First reaction), followed by a labeled oligonucleotide containing a sequence complementary to a part of the amplified fragment, and a microtube in which a substance specific to the label used in the first reaction is immobilized. The step of adding the product of the first reaction to the plate (second reaction), and then measuring the signal generated from the labeling of the labeled oligonucleotide used in the second reaction (third reaction) to detect the presence or absence of the target nucleic acid It is characterized by doing.

As used herein, the term "label" refers to a receptor substance that specifically recognizes and binds to a substance immobilized on a microplate, and includes, for example, biotin, but is not limited thereto. The term “label” refers to a substance that emits a signal so that the behavior of the oligonucleotide can be monitored from the outside, and examples thereof include alkaline phosphatase and peroxidase. When an enzyme is used for labeling, 4-methylumbelliferyl phosphate is used when the label is alkaline phosphatase, and parahydroxyphenylpropionic acid is used when the label is peroxidase.

When the target nucleic acid is RNA, the reaction is carried out by transferring the DNA to DNA using reverse transcriptase.

[0017]

According to the present invention, the presence or absence of a target nucleic acid can be determined with high sensitivity and in a short time without performing electrophoresis.
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.

[0018]

【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. This solution is serially diluted and 0.4fg, 4fg, 40fg, 400fg, 4000f
A g / 3ul TE solution was used for PCR. The PCR reaction conditions were 10 μl of 10 × buffer and 4.8 μl of dNTP (1.
0.75 μl each of primers (a) and (b) having a sequence specific to the Vibrio parahaemolyticus tdh gene
(25 nmol / ml), Nonidet P-40, Tween 20 0.5% each 3u
l, 0.15 µl (5 units / ul) of heat-resistant DNA polymerase was added to prepare 10 µl of a reaction solution. 50 μl of mineral oil (SIGMA) was added to the container containing the reaction solution to prepare a reaction solution. The composition of each buffer is shown below. 10x
Buffer: 500 mM KCl 100 mM Tris HC1 15 mM MgC
l _2, 0.1% gelatin), dNTPs solution: dATP d
CTP dGTP Refers to a mixture of dTTPs. 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 the 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. (a) is a reaction of monomethoxytritylamine linker at the final step of the usual synthesis to give a hexanolamine (amino group) at the 5 'end.
Was combined. After the deprotection operation, purification was performed by high performance liquid chromatography equipped with a C18 reverse phase column. Then (a)
Dissolve 150 ul (1 OD, 5 nmol) in 1X PBS (PH7.4)
M N-hydroxysuccinimide biotin (DMF solution) 150
Reacted with ul. After reacting overnight at room temperature, the mixture was purified by a gel filtration column (G-25, Pharmacia), the fraction was shaken under reduced pressure, purified by reversed-phase high performance liquid chromatography, and used for PCR. After deprotection, the primer (b) was purified by reversed-phase high-performance liquid chromatography. After completion of PCR, 2 ul of each was used for the subsequent reactions.

Second reaction The sample after completion of PCR was taken in 2 ul portions, dissolved in 50 ul of 1 × TE, heated at 95 ° C. for 5 minutes, immediately quenched in ice water, and denatured into single-stranded DNA.

Thereafter, an oligonucleotide (C) labeled with alkaline phosphatase at the 5'-position was added to the well of a 96-well microplate (Nunc) to which streptavidin had been previously fixed. 5 'dCCAAATCACTTTTACTTGG3' 100 pmol / ml
2XSSC, (2.5 XSSC, 1% Tween 20
45 μl of the denatured sample was added to a 96-well microplate to which 30 μl of the solution had been added, 50 μl of 1 × TE, and 50 μl of distilled water, and allowed to stand at room temperature for 15 minutes.

The method of preparing the oligonucleotide labeled with alkaline phosphatase used in the reaction is described in Murakami, Ae
t al, Nucl. Acid Res. 1989.17.5587-5595) and Tada et al.
The method was reported to the 58th Annual Meeting of the Chemical Society of Japan (1989), 3IIG31.

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 capronsan (Sigma, dimethylamide solution) was added thereto, followed by reaction at room temperature for 2-3 hours.

The reaction solution was purified using a G-25 gel filtration column (PD-10, Pharmacia). The solution was dissolved in 1XPBS so that the absorbance at 280 nm became 0.5 OD / ml, and 200 μl of the solution was added to a 96-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. Discard the solution and remove 0.
After washing the wells twice with 1XPBS containing 1% Tween 20, 20
ug / ml streptavidin (BRL) was added to each well and left at room temperature for 2-3 hours. Then 0.5% Tween 20
The wells were adjusted by washing the wells twice with 1XPBS containing.

After standing at room temperature for 15 minutes, the solution in the well was discarded, and the plate was then placed in 1X containing 0.5% Tween 20.
Pre-warmed to 50 ° C twice with 200ul of PBS for 1 minute at room temperature
1 minute in 200ul of 1X PBS containing 0.5% Tween 20 for 1 minute
And once again for 1 minute at room temperature, then 1X PBS
The solution in the wells was replaced with 200 ul and then 200 ul of 0.2 M Tris-HCl buffer (PH9.5) containing 1 mM magnesium chloride.

Third reaction 200 μl of 0.2 M Tris-HCl buffer (PH9.5) containing 0.01 mM 4-methylumbelliferyl phosphate (Sigma) and 1 mM magnesium chloride was added to each well, and the enzyme was added at 37 ° C. for 15 minutes. The reaction was performed. Take 150ul of reaction solution and 400ul of 50mM
EDTA was added to stop the enzyme reaction. The solution was excited with a Shimadzu spectrophotometer (RF5000) at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm.
Was measured. The results are shown in the attached graph. The results of this method are shown in FIG.

As a result, 10 copies or less of the DNA of Vibrio parahaemolyticus WP1 strain was reduced to about 45
It could be detected in a short time (S / N = 2). This result was about 1-2 orders of magnitude higher sensitivity than the conventional electrophoresis method.

In this method, hybridization and 9
Immobilization of the target nucleic acid on the 6-well microplate occurred at the same time, but no adverse effect was observed.

Example 2 In order to examine the specificity of this method, 23 clinical isolates (including Vibrio parahaemolyticus WP1 strain) were used in the same manner as described in Example (1).

Whether or not each strain has the tdh gene has been examined by DNA colony hybridization according to Shirai et al. (1989, Journal of the Bacteriological Society of Japan 44: 196). In addition, tdh gene and high DNA of about 83% and 68%
A thermostable hemotoxin-like hemolytic toxin gene with homology ( tr
h 1 gene, and the presence or absence of trh 2 gene) is also described at the same time.

The presence or absence of the trh gene was examined by DNA colony hybridization according to Kishishita et al. (1990, Journal of the Bacteriological Society of Japan 45: 350).

The results are plotted in FIG. All the tdh- positive strains had a fluorescence intensity (signal) of 140 or more. 1- sided tdh- negative strain ( trh- negative and trh 1 or
In all cases, the fluorescence intensity was 11 or less. This method does not cross-react with the trh gene, which has a very high DNA homology.
The tdh gene-specific detection was possible.

As described above, the present method is applied to Examples (1) and (2).
As shown in Table 2, it was found that the Vibrio parahaemolyticus tdh gene can be specifically detected with high sensitivity and high specificity.

[0035]

According to the present invention, the presence or absence of a target nucleic acid can be detected with high sensitivity without electrophoresis and in a short time of about 45 minutes. In addition, since a tool such as a 96-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.

[Brief description of the drawings]

[Fig. 1] DNA of Vibrio parahaemolyticus WP1 strain
Figure detected by the present invention

FIG. 2 is a diagram showing the DNA of 23 clinical isolates detected according to the present invention.

──────────────────────────────────────────────────の Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C12Q 1/68 G01N 33/50 G01N 33/52 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. a step of using two oligonucleotides labeled at least one of them as a chain length reaction primer to selectively amplify a specific DNA in a target DNA using a DNA synthase (first reaction); Next, a step of adding the product of the first reaction to a microplate in which a labeled oligonucleotide containing a sequence complementary to a part of the amplified fragment and a substance specific to the label used in the first reaction is immobilized (second step) A method for detecting the presence or absence of a target nucleic acid in a step (third reaction) of measuring a signal generated by labeling of the labeled oligonucleotide used in the second reaction.