JP3241496B2 - Amplification and detection of target nucleic acid sequences using thermostable enzymes - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for amplifying a specific nucleic acid sequence and RNA of a specific nucleic acid sequence obtained by the amplification method.
The present invention relates to a method for detecting a nucleic acid sequence of interest from a copy or a DNA copy and reagent kits thereof.

[0002]

2. Description of the Related Art Methods for diagnosing diseases by detecting genes such as DNA or RNA have been developed for detecting bacteria, viruses and the like. In some kinds of specimens, a sufficient amount of nucleic acid exists for direct detection, but it is difficult to directly detect the target gene when the target gene is extremely small or the abundance ratio is very small. Conventionally, the number of target genes has been increased by a cell culture method or a bacterial culture method, but these methods have a drawback that they require a long time.

A polymerase chain reaction (PCR; Japanese Patent Publication No. 4-67957) is known as another method for amplifying a target nucleic acid. In this method, the degree of amplification of the target nucleic acid is adjusted by the number of cycles. The amplification factor is theoretically 2
ⁿ (n is the number of cycles). It took 20-30 cycles to actually amplify the target nucleic acid to a detectable amount.

[0004] As yet another nucleic acid amplification method, a replication R
An NA-based amplification system is known (JP-A-2-586).
4, JP-A-2-500565, JP-A-2-50153
2). These methods include the use of a DNA-dependent RNA polymerase promoter sequence in a primer used when synthesizing double-stranded DNA from a target nucleic acid,
Following double-stranded DNA synthesis, the synthesized double-stranded DNA
With a DNA-dependent RNA polymerase as a template
RNA corresponding to the target nucleic acid is synthesized. Further, a DNA / RNA strand is synthesized from the synthesized RNA by an RNA-dependent DNA polymerase, and the DNA strand is separated to obtain a single-stranded DNA. As a method for separating DNA, a method using heat denaturation (JP-A-2-500565, JP-A-2-50-50)
1532) and a method using ribonuclease H (JP-A-2-5864). The single-stranded D thus obtained
With the NA and the primer, double-stranded DNA containing a promoter sequence of a DNA-dependent RNA polymerase is synthesized to perform an RNA transcription reaction. According to this method, tens to thousands of RNA molecules can be transcribed and amplified from one double-stranded nucleic acid by a DNA-dependent RNA polymerase.
The amplification efficiency per cycle is higher than that of the R method. In addition, when ribonuclease H is used, the temperature cycle required for the PCR method is not required, and amplification can be performed more easily.

[0005]

The amplification efficiency of a replication RNA-based amplification system is high, but the conventional enzymes used in the reaction system, ie, RNA-dependent DNA polymerase, DNA-dependent RNA polymerase, and DNA-dependent DNA polymerase have low thermal stability. Since the temperature is low, the temperature at the time of the amplification reaction cannot be increased, non-specific hybridization between the template nucleic acid and the primer cannot be avoided, and a decrease in specificity becomes a problem. In addition, instability during supply and storage of the enzyme as a reagent becomes a serious problem, and requires frozen storage or refrigerated storage. In the amplification method not using ribonuclease H, heat denaturation is used for denaturation of double-stranded nucleic acid. However, since the enzyme is unstable, it is necessary to sequentially add a group of enzymes each time heating is performed. An object of the present invention is to reduce specificity by non-specific hybridization, supply of enzyme reagents,
The aim is to provide a solution to the instability during storage.

[0006]

That is, the present invention is a method for amplifying a target nucleic acid sequence using a thermostable enzyme, comprising the following steps. Step 1: A first primer having a nucleic acid sequence sufficiently complementary to the nucleic acid sequence of the first template is hybridized to the single-stranded first template nucleic acid obtained by denaturing the target nucleic acid as necessary, and heat-resistant DNA Extending the first primer with a dependent DNA polymerase and / or a thermostable RNA-dependent DNA polymerase to obtain a first primer extension that is a second template complementary to the first template nucleic acid; Step 2: from the first template nucleic acid Separating the first primer extension to obtain a single-stranded second template nucleic acid; Step 3: a nucleic acid sequence complementary to the single-stranded second template nucleic acid with respect to the second template nucleic acid sequence and 5 thereof 'Hybridize a second primer having a promoter sequence at the terminal side, extend with a heat-resistant DNA-dependent DNA polymerase, and extend a second primer extension product complementary to the second template nucleic acid. Producing a double-stranded DNA intermediate having the target nucleic acid sequence bound to a promoter sequence operable in this manner; wherein the first primer is complementary to the target nucleic acid sequence and the second primer is Homologous to the sequence, the 3 'end of the first primer is directed to the 3' end of the second primer on the complementary strand) Step 4: a heat-resistant DNA-dependent RNA capable of recognizing the promoter sequence Using a polymerase, multiple RNAs of the target nucleic acid sequence can be separated from the double-stranded DNA intermediate.
Increasing the copy: and Step 5: Repeat Steps 1 to 4 as necessary as necessary, using the RNA copy as the first template nucleic acid.

[0007] The present invention also relates to a single-stranded RNA, a double-stranded DNA or a DN which is an amplification product of a target nucleic acid by the above-mentioned amplification method.
A method for detecting a target nucleic acid sequence, which comprises subjecting an A / RNA hybrid to a denaturation treatment if necessary, hybridizing a labeled probe, and detecting a label of a hybridized labeled probe or a label of a non-hybridized labeled probe. It is.

Further, the present invention provides a reagent kit for amplifying a target nucleic acid sequence, comprising: (a) a first primer having a nucleic acid sequence sufficiently complementary to a nucleic acid sequence of a first template;
(b) a nucleic acid sequence complementary to the nucleic acid sequence of the second template and a second primer having a promoter sequence at its 5 ′ end, (c) a thermostable RNA-dependent DNA polymerase,
(d) a thermostable DNA-dependent RNA polymerase, (e) a thermostable DNA-dependent DNA polymerase, (f) ribonucleoside triphosphate and (g) deoxyribonucleoside triphosphate, provided that the first primer is complementary to the target nucleic acid sequence. Wherein the second primer is homologous to the target nucleic acid sequence and the 3 'end of the first primer is directed to the 3' end of the second primer on the complementary strand. Is a reagent kit for amplifying a target nucleic acid sequence.

The present invention also relates to a reagent kit for amplifying a target nucleic acid sequence, comprising: (a) a nucleic acid sequence sufficiently complementary to a nucleic acid sequence of a first template and a promoter sequence at the 5 ′ end thereof. (B) a second primer having a nucleic acid sequence complementary to the nucleic acid sequence of the second template and a promoter sequence at the 5 ′ end thereof, (c) a thermostable RNA-dependent DNA polymerase, (d ) Heat resistant DN
A-dependent RNA polymerase, (e) a thermostable DNA-dependent DNA polymerase, (f) ribonucleoside triphosphate and (g) deoxyribonucleoside triphosphate, provided that it is complementary to the first primer target nucleic acid sequence; A target using a thermostable enzyme, wherein the second primer is homologous to the target nucleic acid sequence and the 3 'end of the first primer is directed to the 3' end of the second primer on the complementary strand. It is a reagent kit for amplifying a nucleic acid sequence.

[0010] Even if the target nucleic acid of the present invention is DNA, it may be RN.
A may be used. In the case of a double strand or a single strand having a higher-order structure, it is subjected to amplification reaction as a single strand in advance by heating, denaturing treatment with an acid, an alkali or the like.

The first primer used in the present invention has a nucleic acid sequence sufficiently complementary to the nucleic acid sequence of the first template which is the target nucleic acid sequence. 3 'of the first primer
The end is directed to the 3 'end of the second primer on the complementary strand. Further, the second primer in the present invention has a nucleic acid sequence complementary to the nucleic acid sequence of the second template and a promoter sequence at the 5 ′ end thereof, and is sufficiently homologous to the target nucleic acid sequence. The first primer has a nucleic acid sequence complementary to the nucleic acid sequence of the first template and, if necessary, 5 ′
It may have a promoter sequence on the terminal side. When the first primer has a promoter sequence, the promoters of the first primer and the second primer may be different or the same. If they are different, heat-resistant DNA-dependent R acting on each promoter
A plurality of NA polymerases are used if necessary. Depending on the type of promoter, it is also possible to select one type of thermostable DNA-dependent RNA polymerase so that both types of promoter function. Amplification efficiency can be further improved by providing both the first primer and the second primer with a promoter function.

The promoter sequence used in the present invention is not particularly limited, but must be a sequence that functions so that a thermostable DNA-dependent RNA polymerase acts. As such a promoter sequence, for example, if the DNA-dependent RNA polymerase of Thermus thermphilus, the 5'-TTCGCGCCCATCGTACACCGAGGCGGTATCCTC-3 '5'-CTTGACGGAGGCGGACGGCGCTGGTACACT-3'5'-CTGGACAGGGCCCCCGTGTCCCGCTATCCT-TCGGTCTCGG 3 '5'-CTTGACCCCGCAGGCCTCGAGGGCTTACCT-3' is known.

[0013] In addition, there are the following. 5'-CTTGACGCCGCCCAGGGCGGGCCTCTACCCT-3 '5'-TTTGAGGGCCTGGGGCAGTACCTCTTCT-3' 5'-TTTGTAAAGTGCTTTATTTCACAAAACT-3 '5'-TTTCACAAAACTGTCCCTCCCCCCGGGTTAGACT-3'5'-3 '-CTTGACAAAAAGGAGGGGGATTGATAGCAT-3' 5'-CGTGAGGGCCACGGCGAGCGCGCCTAGGGGT-3 '5'-CTAGTCCAAGGGAAAGTATAGCCCAAGGTACACT-3' 5'-CTTGACGTGAAACTTGAAGACCACCATCTCAA-3 '

In general, phage promoters have high specificity, but promoters of other organisms may not always be highly specific. The term “high specificity” as used herein means that a promoter sequence capable of acting on a promoter-dependent DNA-dependent RNA polymerase has its DNA
It means that one or more dependent RNA polymerases are very low and that the activity as a promoter is very low. Accordingly, it is shown that even if various promoters are present in the sample nucleic acid to be detected, the promoter-dependent DNA-dependent RNA polymerase can act with practically no problem.

On the other hand, in bacteria and other organisms, DNA-dependent R
It is known that there is not always one type of promoter sequence on which NA polymerase acts, and there are a plurality of types of promoter sequences. Some bacteria and fungi have high commonality. Therefore, it is conceivable that the sample nucleic acid to be detected may contain a promoter sequence on which the DNA-dependent RNA polymerase to be used acts.

In the present invention, as a result of studying in consideration of such points, the reaction at 50 to 70 ° C. at which a thermostable DNA-dependent RNA polymerase can act does not result in the expression of a non-specific promoter function. Highly specific amplification and detection are possible.

Oligonucleotides serving as the first primer and the second primer are, for example, ABI (Applied Bi
osystems Inc.) using a DNA synthesizer model 391 by the phosphoramidite method. Other synthesis methods include the phosphoric acid triester method, the H-phosphonate method, and the thiophosphite method. It may also be isolated from biological sources, for example from restriction endonuclease digests. There is no particular limitation on the length and structure of the nucleic acid as long as it is set to function as a primer. Generally, the portion complementary to the first template or the second template of the first primer and the second primer is 6 to 50 nucleotides, preferably 1 to 50 nucleotides.
0-30 nucleotides. It is also possible to provide a spacer between the promoter region and the primer region that hybridizes with the target nucleic acid, but this spacer is generally 0 to 100 nucleotides, preferably 0 to 2 nucleotides.
0 nucleotides are preferred.

The term "thermostable enzyme" as used in the present invention means that an enzyme reaction can be carried out at 50 to 70 ° C. in order to specifically carry out hybridization, and 90 to 9 for heat denaturation of nucleic acid.
It refers to an enzyme with little deactivation even at 5 ° C. for about 10 seconds to 10 minutes. In addition, such enzymes are generally sufficiently stable even under refrigerated storage or room temperature storage, and in many cases, there is no need for frozen storage, and the stability during supply and storage is also good.

As the thermostable RNA-dependent DNA polymerase (also referred to as thermostable reverse transcriptase), DNA-dependent DNA polymerases derived from Thermus thermophilus and Thermus aquaticus have an activity. Are known. Heat resistance D
Examples of NA-dependent RNA polymerase (also referred to as thermostable RNA polymerase) include Thermus thermophilus (Thermal Thermofils).
rmus thermophilus). The heat resistance D
NA-dependent RNA polymerase can recognize a promoter sequence. Examples of the thermostable DNA-dependent DNA polymerase (also referred to as thermostable DNA polymerase) include Thermus thermophilus, Thermus aquaticus, Pyrococcus furiosus, and Thermococcus litoralis. ), From Thermus flavus.

In step 1 of the present invention, a first primer is hybridized to a single-stranded first template nucleic acid which has been denatured as required, and a heat-resistant DNA-dependent DNA polymerase and / or a heat-resistant DNA polymerase are added in the presence of deoxyribonucleoside triphosphate. It is extended by a sex RNA-dependent DNA polymerase to obtain a first primer extension which is a second template complementary to the first template nucleic acid. The first primer extension is
When the first template is RNA, heat-resistant RNA-dependent DNA
Polymerase, first primer extension, the first template is D
In the case of NA, thermostable DNA-dependent DNA polymerase, when the target nucleic acid sequence contains DNA and RNA,
Thermostable DNA-dependent DNA polymerase and thermostable RN
Extension by A-dependent DNA polymerase yields a DNA extension product.

In general, RNA-dependent DNA polymerases have a DNA-dependent DNA polymerase activity, and DNA-dependent DNA polymerases derived from Thermus thermophilus and Thermus aquaticus have RNA-dependent DNA polymerase activities.
It is known that a dependent DNA polymerase activity exists, and it is also possible to use one kind of DNA polymerase having both activities in common.

In step 2 of the present invention, the extension product from the first template is separated from the first template nucleic acid to obtain a single-stranded second template nucleic acid. The separation is performed by a method such as acid, alari, or heating. In particular, it is preferable to separate the second template nucleic acid from the first template nucleic acid by heat denaturation.

In step 3 of the present invention, a single-stranded second template nucleic acid is hybridized with a second primer having a promoter sequence at its 5 'end, and a heat-resistant DNA-dependent DNA
It is extended by a polymerase to obtain a second primer extension complementary to the second template nucleic acid. The second primer extension is a double stranded DNA intermediate having the target nucleic acid sequence linked to a functional promoter sequence. Alternatively, a primer having a promoter sequence at the 5 'end may be used as a first promoter complementary to the first template in step 1. The thermostable DNA polymerase used in step 1 is DNA
When it has a dependent DNA polymerase activity, it can be shared with the thermostable DNA-dependent DNA polymerase in the present step 3.

In step 4 of the present invention, a large number of RNA copies of the target sequence are increased from the double-stranded DNA intermediate obtained in step 3 using a heat-resistant DNA-dependent RNA polymerase capable of recognizing a promoter sequence. .

In step 5 of the present invention, if necessary, the above steps 1 to 4 are repeated as many times as necessary using the RNA copy obtained in step 4 as the first template nucleic acid, so that the DNA having the target nucleic acid sequence and / or Alternatively, RNA can be amplified.

In the amplification method of the present invention, it is preferable to carry out the above steps 1 to 5 without successively adding reagents. The hybridization temperature of the present invention is 50 to 80 ° C, preferably 50 to 70 ° C, and a temperature at which three kinds of thermostable nucleic acid polymerases react efficiently is selected.

That is, the present invention uses a heat-resistant enzyme for all of the RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and DNA-dependent RNA polymerase required for a replication RNA-based amplification system, thereby allowing the amplification reaction of a target nucleic acid to be a nucleic acid template. And high temperatures sufficient to avoid non-specific hybridization between the primers and primers. As a result, non-specific amplification does not occur, and highly specific amplification becomes possible. The thermostable enzyme is stable not only at a low temperature but also at a normal temperature. There is almost no decrease in the activity at the time of supply and at the time of storage, and the instability can be eliminated. Even when denaturation is performed by heating without using ribonuclease H, the use of a thermostable enzyme prevents inactivation of a group of enzymes and enables amplification without successively adding enzymes.

In the present invention, the reaction temperature is adjusted to 50 to 70 by using a thermostable DNA-dependent RNA polymerase.
The above-mentioned problem can be overcome by setting the temperature to ° C. and performing the reaction under the condition that the non-specific promoter function is not expressed.

The reagent used in the amplification reaction of the present invention is preferably set so that a plurality of enzymes are reacted simultaneously. Specifically, an appropriate amount of (a) a first primer having a sequence sufficiently complementary to the nucleic acid sequence of the first template and, if necessary, a promoter sequence at the 5 ′ end thereof, and (b) a second template (C) a thermostable RNA-dependent DNA polymerase; (d) a thermostable DNA-dependent RNA polymerase; e) a thermostable DNA-dependent DNA polymerase, and (f)
Ribonucleoside triphosphate and (g) deoxyribonucleoside triphosphate are essential components.

The concentration of each of the first primer and the second primer is generally 10 to 5000 nM, preferably 100 to 500 nM.
~ 500 nM. As the concentration of ribonucleoside triphosphate and deoxyribonucleoside triphosphate, generally, dNTP 10 to 10,000 uM, rNTP 10 to 10,000 uM
, Preferably dNTP 100-2000 uM, rNTP 100-4000 uM
It is. The concentration ratio of ribonucleoside triphosphate and deoxyribonucleoside triphosphate is generally 1/10 to 2/1, preferably 1/2 to 3/4.

As the thermostable RNA-dependent DNA polymerase, Thermus thermophilu
s), It is known that the thermostable DNA-dependent DNA polymerase of Thermus aquaticus has its activity, and the RNA-dependent DNA polymerase activity is expressed by selecting the type and amount of divalent metal ions. It is known to However, it is not easy to simultaneously express RNA-dependent DNA polymerase and DNA-dependent DNA polymerase activities, and the present inventors have conducted intensive studies on the conditions for maximizing the expression of the two enzyme activities. The ratio is 1: 1 to 4: 1, preferably 1.
It has been found that a condition of 5: 1 to 3: 1 is preferable. Further DN
The type and amount of the divalent metal ion are also important for the A-dependent RNA polymerase, and in order to maximize the activity of these trivalent metal ions, the ratio of Mg ion to Mn ion should be 1: 1 to 4:
1, preferably 1.5: 1 to 3: 1 and dNTP: rNTP = 1: 10
~ 10: 1, preferably 1: 2 to 2: 1.

Although the thermostable enzyme itself has been known, it has not been known to use a plurality of thermostable enzymes in combination as in the present invention. Further, the nucleic acid amplification reaction does not occur even if the enzyme is simply combined with a thermostable enzyme, and it is not easy to achieve higher amplification efficiency by cycling.

In the present invention, the reaction time depends on the nucleic acid sequence to be amplified,
It varies depending on various conditions such as the primer sequence, Tm, and enzyme amount. The reaction time for one cycle is about 5 to 300 minutes, preferably about 20 to 60 minutes, and the number of cycles is 0 (no repetition) to 100, preferably 3 10 to 10 times is appropriate. These conditions are set in consideration of amplification efficiency, amplification amount, and reaction time. After performing the enzyme reaction at about 50 to 70 ° C, the reaction solution is raised to about 90 to 95 ° C to dissociate the double strand of the generated nucleic acid into a single strand, and the temperature is again raised to 50 to 70 ° C.
New primers are bound, extended, and amplified. As a result, a large number of nucleic acid copies having the target nucleic acid sequence can be generated. Further, by repeating this (cycle), larger amplification can be performed.

The nucleic acid amplified by the amplification method of the present invention can be detected as required. Detection of the amplified nucleic acid can be performed by measuring RNA copy, measuring double-stranded DNA having a promoter sequence,
/ RNA hybrids can also be measured. These can be detected by a general measuring method. Elongation reaction by nucleic acid polymerase, at the time of transcription reaction, using a labeled substance such as ³² P, biotinylated nucleotides as riboxynucleoside triphosphate or deoxyribonucleoside triphosphate to be added, incorporating the label into the amplified product, Measure the label. In addition, a method for measuring the label in the amplification product using a labeled primer,
There is a method of detecting an amplified nucleic acid using a labeled probe. Specific detection methods include fractionation by electrophoresis, Southern blot, Northern blot, dot blot, slot blot, and sandwich hybridization. It is also possible to obtain the concentration in the test sample by performing a quantitative measurement. In addition, the quantitative property can be improved by a method using an internal standard (Japanese Patent Application Laid-Open No. 62-205800).

In the detection method using a labeled primer or a labeled probe, a known labeling substance such as a radioisotope, an enzyme, a fluorescent substance or a luminescent substance can be used as the labeling substance. Further, it is possible to prepare reagent kits in which the reagents used in the present invention are prepared.

[0036]

Next, the present invention will be described with reference to examples. Reference Example 1 Synthesis of Various Oligonucleotides Using ABI DNA synthesizer Model 391, various oligonucleotides having the following sequences were synthesized by the phosphoramidite method. The procedure was performed on a 0.2 μM scale according to the manual of ABI. Deprotection of the oligonucleotide was performed with aqueous ammonia at 55 ° C. for 15 to 18 hours. Purification was performed on a reverse phase column with FPLC manufactured by Hitachi, Ltd. Oligonucleotide with a promoter region (1): This oligonucleotide is a promoter region linked by a linker, the gene of Vibrio parahaemolyticus thermostable toxin (VP-TDH) gene.
It consists of a region complementary to the nucleotide sequence at positions 313 to 326 (Sequence Table 1). Oligonucleotide (2): 179 to 20 of VP-TDH gene
Second homologous oligonucleotide (24mer) (Sequence Table 2). Orido nucleotide (3): 254 to 27 of VP-TDH gene
Seventh complementary oligonucleotide (24mer) (Sequence Table 3). The phosphate group at the 5 'end is labeled with ³² P.

Example 1 Amplification Reaction Using Oligonucleotide (1) Having Oligonucleotide Promoter Region and Oligonucleotide (2) Amplification Reaction of Vibrio parahaemolyticus Genome Using Oligonucleotide (1) and Oligonucleotide (2) of Reference Example 1 Was performed to obtain multiple RNA copies. The reaction conditions are shown below. Reaction solution 10 mM Tris-HCl pH8.3 75 mM KCl 6 mM MgCl ₂ 3 mM MnCl ₂ 0.6 mM dNTP 0.4 mM rNTP 20 pmol oligonucleotide (1) 20 pmol oligonucleotide (2) Tth-DNA polymerase 60 units Tth-RNA polymerase 2.5 units Vibrio parahaemolyticus ( TDH toxin-producing strain) Genome 1 pg Reaction: 94 ° C, 1 minute 70 ° C, 10 minutes Then (1) 94 ° C, 1 minute (2) 60 ° C, 30 minutes (3) 70 ° C, 30 minutes (1), (2) ) And (3) are repeated 5 times.

Example 2 Measurement of Amplified Nucleic Acid The nucleic acid amplified in Example 1 was diluted, and the nucleic acid was denatured with 0.3 N NaOH. Then, 50 μl of the nylon membrane, Hybond N ⁺ (A
(manufactured by mersham). As a control, the nucleic acid of Vibrio parahaemolyticus (VP) was denatured with 0.3 N NaOH, and then 50 μl was dot-blotted. Nylon film 6
× SSC (1 × SSC means 0.15 M NaCl, 0.015 M sodium citrate (pH 7.0)), 5 × Danehart solution (1 × Denhart solution is 0.02% ficoll, 0.02% polyvinylpyrroline, 0.02% % Bovine serum albumin) in 100 μl of 1 mM EDTA, 10 μg of boiled salmon semen DNA (average 500 bases).
After prehybridization at 1 ° C. for 1 hour, the oligomer (3) prepared in Reference Example 1 was added to the above solution, followed by hybridization at 55 ° C. for 1 hour. After sufficiently washing the nylon membrane in 6 × SSC at 55 ° C., it was dried. X-ray film (New AI
F RX (manufactured by Fuji Photo Industry Co., Ltd.) and exposed at -80 ° C for 24 hours. When the photosensitivity of the film was quantified synthetic RNA, approximately 10 ⁵ times that the genomic nucleic acid in the control of VP R
NA was synthesized. As a result, it was shown that the amplification method of the present invention was effective. Further, it was proved that the amplification method of the present invention proceeded without requiring addition of a new enzyme every time the temperature cycle was performed.

Example 3 Amplification was carried out in the same manner as in Example 1 using about 1 g of nucleic acid purified from about 0.5 g of stool of a patient suspected of food poisoning by proteinase K treatment, phenol / chloroform extraction and ethanol precipitation as a template. Hybridization was performed in the same manner as in Example 2. As a control, Vibrio parahaemolyticus was separated using a TCBS separation medium. TDH toxin was detected by ELISA (manufactured by Nissui Pharmaceutical) for vibrio parahaemolyticus grown. The results are shown in the table below.

[0040]

[Table 1] As shown in the above table, good results were obtained which agreed well with the conventional method.

[0041]

Industrial Applicability The present invention is a nucleic acid amplification method having high amplification efficiency and high specificity because a thermostable enzyme having higher stability is used as compared with the conventional replication RNA-based amplification method. Since a group of thermostable enzymes is used in the method of the present invention, there is no inactivation of the group of enzymes during heat denaturation, and there is no need to add enzymes sequentially. The conventional nucleic acid amplification method works at room temperature, but when separating the second template nucleic acid from the first template nucleic acid, it needs to be added a new group of enzymes in order to perform amplification again because it is inactivated by heating. However, in the present invention, since a group of thermostable enzymes is used, no inactivation of the enzyme is observed and no new addition is required. Since the amplification can be performed with the reaction container sealed, the contamination which is a major problem in the amplification method can be reduced.

[0042]

[Sequence list]

SEQ ID NO: 1 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA Sequence characteristics Location: 1..24 Other characteristics: 313 to 326 of Vibrio parahaemolyticus TDH gene
It has a sequence complementary to the th sequence. Array GACACCGCTG CCATTGTATA GTCT 24

SEQ ID NO: 2 Sequence length: 54 Sequence type: Number of nucleic acid chains: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbol: Promoter location : 1..30 Method for determining the characteristics: S Other characteristics: Promoter sequence Location: 31..54 Other characteristics: From position 179 to 202 of Vibrio parahaemolyticus TDH gene
It has a sequence homologous to the second sequence. Sequence CTTGACAAAA AGGAGGGGGA TTGATAGCAT CTGACTTTTG GACAAACCGT AATG 54

SEQ ID NO: 3 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1..24, etc. Features: Vibrio parahaemolyticus TDH gene 254 to 277
It has a sequence complementary to the th sequence. Sequence CAGGTACTAA ATGGTTGACA TCCT 24

[Brief description of the drawings]

FIG. 1 illustrates the reaction of the present invention.

[Explanation of symbols]

 1: First template 2: First primer 3: Second template 4: Second primer 5: Functional promoter sequence 6: Double-stranded DNA intermediate 7: RNA copy

──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-2-5864 (JP, A) JP-A-4-507197 (JP, A) International Publication 92/8800 (WO, A1) Biochemistry, 30 (1991) p. 7661-7666 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/09 C12Q 1/68 BIOSIS (DIALOG) MEDLINE (STN)

Claims (9)

(57) [Claims] 1. The method according to claim 1 , further comprising the steps of:
In the method of amplifying a target nucleic acid sequence using a thermophilic enzyme,
The ratio of Mg ion to Mn ion in the liquid is from 1: 1 to 4:
1 and the ratio between dNTP and rNTP is 1:10
Using a thermostable enzyme characterized by being set at 10: 1
A method for amplifying a target nucleic acid sequence. Step 1: A first primer having a nucleic acid sequence sufficiently complementary to the nucleic acid sequence of the first template is hybridized to the single-stranded first template nucleic acid obtained by denaturing the target nucleic acid as necessary, and heat-resistant DNA Dependent DN
A polymerase and / or thermostable RNA-dependent DNA
Extending the first primer with a polymerase to obtain a first primer extension, which is a second template complementary to the first template nucleic acid; Step 2: separating the first primer extension from the first template nucleic acid into one Obtaining a second template nucleic acid of strand; Step 3:
A single-stranded second template nucleic acid is hybridized with a nucleic acid sequence complementary to the nucleic acid sequence of the second template and a second primer having a promoter sequence at the 5 ′ end thereof, and the mixture is subjected to heat-resistant DNA-dependent DNA polymerase. Extending to obtain a second primer extension complementary to the second template nucleic acid, thus producing a double-stranded DNA intermediate having the target nucleic acid sequence linked to a functional promoter sequence; One primer is complementary to the target nucleic acid sequence, the second primer is homologous to the target nucleic acid sequence, and the 3 'end of the first primer is directed to the 3' end of the second primer on the complementary strand ) Step 4: Using a heat-resistant DNA-dependent RNA polymerase capable of recognizing the promoter sequence, a large number of Rs of the target nucleic acid sequence are converted from the double-stranded DNA intermediate. Increasing the A copy: The Step 5: required by repeating a necessary number of times steps 4 from the step 1 by using the RNA copy as a first template nucleic acid. 2. The method for amplifying a target nucleic acid sequence using a thermostable enzyme according to claim 1, wherein the second template nucleic acid is separated from the first template nucleic acid by heat denaturation. 3. The method according to claim 1, wherein the first primer having a nucleic acid sequence complementary to the nucleic acid sequence of the first template has a promoter sequence at the 5 ′ end. A method for amplifying a target nucleic acid sequence using a thermostable enzyme. 4. A thermostable RNA-dependent DNA polymerase, DNA-dependent DN derived from Thermus thermophilus
4. The method for amplifying a target nucleic acid sequence using a thermostable enzyme according to claim 1, wherein the RNA-dependent DNA polymerase activity of A polymerase is used. 5. The method for amplifying a target nucleic acid sequence using a thermostable enzyme according to claim 1, wherein the thermostable DNA-dependent RNA polymerase is derived from Thermos thermophilus. 6. The method for amplifying a target nucleic acid sequence using a thermostable enzyme according to claim 1, wherein the thermostable DNA-dependent DNA polymerase is derived from Thermus thermophilus. 7. A single-stranded RNA, double-stranded DNA or DN which is an amplification product of the target nucleic acid sequence according to claim 1.
A method for detecting a target nucleic acid sequence, which comprises subjecting an A / RNA hybrid to a denaturation treatment if necessary, hybridizing a labeled probe, and detecting a label of a hybridized labeled probe or a label of a non-hybridized labeled probe. . 8. The method according to claim 1, wherein the target nucleic acid sequence is increased without sequentially adding reagents.
A reagent kit for spreading, comprising : (a) a first primer having a nucleic acid sequence sufficiently complementary to the nucleic acid sequence of the first template; (b) a complementary primer to the nucleic acid sequence of the second template. A second primer having a nucleic acid sequence and a promoter sequence at the 5 ′ end thereof, (c) a thermostable RNA-dependent DNA polymerase, (d) a thermostable DNA-dependent RNA polymerase, (e) a thermostable DNA-dependent DNA polymerase,
(F) ribonucleoside triphosphate and (g)
And deoxyribonucleoside triphosphate,
However, the first primer is complementary to the target nucleic acid sequence,
The second primer is homologous to the target nucleic acid sequence,
The 3 'end of the first primer is directed to the 3' end of the second primer on the complementary strand and is further included in the reaction solution.
And the ratio of Mg ions to Mn ions is 1: 1 to 4: 1
And the ratio of dNTP to rNTP is 1:10 to 1
A reagent kit for amplifying a target nucleic acid sequence using a thermotolerant enzyme, which is set to be 0: 1 . 9. A method for increasing a target nucleic acid sequence without successively adding reagents.
A reagent kit for amplification, comprising : (a) a nucleic acid sequence sufficiently complementary to the nucleic acid sequence of the first template and its 5 ′
A first primer having a promoter sequence on the terminal side,
(B) a nucleic acid sequence complementary to the nucleic acid sequence of the second template and a second primer having a promoter sequence at its 5 'end, (c) a thermostable RNA-dependent DNA polymerase, (d) a thermostable DNA-dependent RNA Polymerase,
(E) a thermostable DNA-dependent DNA polymerase, (f) ribonucleoside triphosphate and (g) deoxyribonucleoside triphosphate, provided that it is complementary to the first primer target nucleic acid sequence and the second primer is the target nucleic acid sequence. Homologous to the sequence, the 3 'end of the first primer is directed to the 3' end of the second primer on the complementary strand , and
The ratio of Mg ion to Mn ion is 1: 1 to 4: 1,
And the ratio between dNTP and rNTP is 1:10 to 10:
1. A reagent kit for amplifying a target nucleic acid sequence using a thermostable enzyme, wherein the kit is set to 1 .