JP7218558B2 - Nucleic acid amplification method and nucleic acid amplification reagent - Google Patents

Description

The present invention relates to methods and reagents for amplifying target nucleic acids expected to be contained in genetic mixtures such as DNA and RNA. Furthermore, the present invention relates to methods and detection reagents for amplifying and qualitatively or quantitatively detecting said target nucleic acid. INDUSTRIAL APPLICABILITY The present invention is useful in the field of clinical diagnosis such as genetic diagnosis, and can amplify and detect target nucleic acids directly from specimens. In this case, it is also useful for rapid and highly sensitive identification of the microorganism and detection of drug resistance genes.

In genetic testing used in clinical diagnosis, the amount of target nucleic acid contained in clinical samples is often very small. It achieves good reproducible measurements.

Examples of the method for amplifying the target nucleic acid include PCR (polymerase chain reaction) method. This method consists of heat denaturation, annealing, and extension reactions using a set of primers consisting of primers complementary and homologous to both ends of a specific DNA sequence in the target nucleic acid, and a thermostable DNA polymerase. By repeating the cycle of steps, a polynucleotide containing the specific DNA sequence can be amplified. On the other hand, when the target nucleic acid is RNA, it is necessary to synthesize cDNA from RNA as a template using reverse transcriptase before performing the PCR method. That is, the procedure is complicated because it requires two steps, a cDNA synthesis step and a PCR reaction step. Moreover, the PCR method requires a special incubator because it requires rapid temperature rise and fall, and is not easy to apply to automation for the purpose of large-scale processing. Furthermore, since it is necessary to repeat the three-step cycle consisting of heat denaturation, annealing, and elongation reaction, there is a limit to speeding up the method. As a method for easily amplifying a specific double-stranded DNA from a target RNA using a reverse transcription reaction and a PCR reaction, Patent Document 1 discloses a 3′→5′ exo A method is disclosed for obtaining double-stranded DNA using a DNA polymerase that lacks nuclease and strand displacement activity.

On the other hand, as a simple amplification method of target RNA, NASBA (Nucleic Acid Sequence-Based Amplification) method (Patent Documents 2 and 3, Non-Patent Document 1), TMA (Transcription Mediated Amplification) method (Patent Document 4), TRC (Transcription -Reverse transcription concerted reaction method (Patent Document 5 and Non-Patent Document 2) and the like have been reported. These methods use a primer containing a promoter sequence for a target RNA, reverse transcriptase and ribonuclease H (RNase H) to generate a double-stranded DNA containing a promoter sequence, and an RNA polymerase to generate a specific base sequence. A chain reaction is then performed using the generated RNA as a template for synthesizing a double-stranded DNA containing the promoter sequence. When amplifying RNA by these methods, an RNA-dependent DNA polymerase activity that synthesizes a complementary DNA strand to the RNA, an RNase H activity that degrades the RNA of the RNA-cDNA duplex, and a DNA that synthesizes a complementary DNA strand to the cDNA. AMV reverse transcriptase, which has a dependent DNA polymerase activity, is commonly used. These amplification methods are easy to apply to automation because they allow RNA amplification under a constant temperature of relatively low temperature (eg, 40° C. to 50° C.). Furthermore, by adding an enzyme having strand displacement activity, DNA amplification at a relatively low constant temperature is possible (Patent Document 6). In Patent Document 6, 96-7 DNA polymerase is used as the enzyme having strand displacement activity.

Detection of the target nucleic acid amplified by the method described above is usually carried out by contacting a probe labeled with a fluorescent dye that can specifically bind to a portion of the target nucleic acid, and detecting the fluorescence derived from the probe bound to the amplification product. and do it. In this case, a large fluorescence intensity ratio, which is also called an S/N ratio (signal-to-noise ratio), is preferable in terms of accurate detection. Also, depending on the measurement item, the faster the detection time, the better. However, in the detection of the target nucleic acid, the improvement of the S/N ratio does not necessarily correlate with the detection time. This is probably because the faster the detection time, the more the amplification products are used in the chain reaction, and the less the fluorescence intensity increases. Therefore, when detecting a target nucleic acid with high accuracy, not only the speed of detection but also the size of the fluorescence intensity ratio is important.

Real-time PCR is known as a method for quantifying target nucleic acids contained in a sample. However, the efficiency and accuracy of amplification by PCR are influenced by various factors such as the treatment temperature, the base sequence of the nucleic acid, the concentrations and types of reaction solution components, and the like. Therefore, it has been difficult to accurately determine the target nucleic acid amount (concentration) by densitometry of the final PCR product or real-time measurement during the PCR process.

In recent years, digital PCR has been developed as a technique for solving this problem and accurately quantifying minute amounts of target DNA (Non-Patent Document 3). In digital PCR, a solution containing target DNA is distributed to a number of microcompartments and PCR is performed in parallel in each individual microcompartment. Some microcompartments contain one or more target molecules, and some microcompartments do not contain any target molecules. The concentration of target DNA can be quantified. As a method for accurately quantifying a trace amount of target RNA, cDNA is synthesized in advance using reverse transcriptase, and then the above-mentioned digital PCR is used, or digital NASBA (Patent Document 7), which replaces the amplification method with the NASBA method. , Non-Patent Document 3).

When detecting an amplification product of a target nucleic acid obtained by digital nucleic acid amplification such as digital PCR or digital NASBA, it is determined as positive or negative based on the fluorescence intensity (signal) of each minute compartment. Therefore, the setting of the reaction conditions and the threshold value of the signal value for judging positive and negative at the time of detection is an issue. Therefore, even when nucleic acids are amplified and detected digitally, the S/N ratio is important in order to detect a very small amount of specific RNA sequences present in a sample with high sensitivity and without false positives.

WO2010/131645 JP-A-2-005864 Japanese Patent Publication No. 4-503451 Japanese Patent Publication No. 4-500759 Japanese Patent Application Laid-Open No. 2000-014400 JP 2015-116136 A U.S. Publication No. 2014/0141502

de Baar, M.; P. et. al, Journal of Clinical Microbiology, 39, 1895-1902 (2001) Ishiguro, T.; et al, Analytical Biochemistry, 314, 77-86 (2003) Vogelstein, B.; et al, Proc. Natl. Acad. Sci. U.S.A. S. A. , 96, 9236-9241 (1999)

The subject of the present invention is a method for amplifying and detecting a very small amount of target nucleic acid present in a sample more efficiently and reproducibly (highly accurately) in a method for amplifying and detecting a target nucleic acid, and the method. To provide a reagent using

The inventors of the present invention have completed the present invention as a result of earnest research to solve the above problems.

That is, aspects of the present invention can be exemplified as follows.

[1]
(i) an enzyme having RNA-dependent DNA polymerase activity;
(ii) an enzyme having DNA-dependent DNA polymerase activity;
(iii) an enzyme having ribonuclease H (RNase H) activity;
(iv) an enzyme having RNA polymerase activity;
(v) a first primer having a sequence complementary to a portion of the target nucleic acid; and (vi) a second primer having a sequence homologous to a portion of the target nucleic acid. , (v) either the first primer or (vi) the second primer has (iv) a promoter sequence for an enzyme having RNA polymerase activity added to its 5′ end, ,
(vii) the amplification reagent further comprising an enzyme having DNA-dependent DNA polymerase activity without 3′→5′ exonuclease activity and with 5′→3′ exonuclease activity.

[2]
(ii) The amplification reagent according to [1], wherein the enzyme having DNA-dependent DNA polymerase activity is an enzyme having no 5′→3′ exonuclease activity.

[3]
(i) the enzyme having RNA-dependent DNA polymerase activity, (ii) the enzyme having DNA-dependent DNA polymerase activity, and (iii) the enzyme having RNase H activity is AMV reverse transcriptase [1] or [ 2].

[4]
The amplification reagent according to any one of [1] to [3], further comprising a strand displacement enzyme.

[5]
A third primer having a sequence homologous to a portion of the target nucleic acid located on the 5'-end side of the target nucleic acid is added to the primer having the promoter sequence of an enzyme having RNA polymerase activity added to the 5'-end side. The amplification reagent according to any one of [1] to [4], further comprising:

[6]
(vii) the enzyme having a DNA-dependent DNA polymerase activity that does not have 3′→5′ exonuclease activity and has 5′→3′ exonuclease activity is Bst DNA polymerase (Full Length) or Tth DNA polymerase; The amplification reagent according to any one of [1] to [5].

[7]
The target comprising the amplification reagent according to any one of [1] to [6], and an oligonucleotide probe whose fluorescence properties change when forming a complementary double strand with a part of the target nucleic acid compared to before formation. Nucleic acid detection reagent.

[8]
A method for amplifying a target nucleic acid, comprising the following steps (1) to (7) (provided that either the first primer or the second primer has an enzyme having RNA polymerase activity on its 5′ end side) of the promoter sequence is added).
(1) Synthesizing cDNA complementary to the target RNA from the target RNA using a first primer having a sequence complementary to a portion of the target RNA and an enzyme having RNA-dependent DNA polymerase activity (2) ) degrading the RNA of the RNA-DNA duplex using an enzyme having ribonuclease H (RNase H) activity; (3) a second primer having a sequence homologous to a portion of the target RNA; Using a DNA-dependent DNA polymerase that does not have →5′ exonuclease activity and has 5′→3′ exonuclease activity, from the single-stranded DNA obtained in step (2) above, the 5′ end is Step (4) of synthesizing a double-stranded DNA having a promoter sequence of an enzyme having RNA polymerase activity added to the side thereof (4) Using an enzyme having RNA polymerase activity corresponding to the promoter sequence, the double-stranded DNA obtained in (3) is synthesized. Step (5) of synthesizing an RNA transcript from the main strand DNA using a primer having a sequence complementary to a part of the RNA transcript synthesized in the step (4) and an RNA-dependent DNA polymerase activity, the step of synthesizing cDNA complementary to the RNA transcript of (4);
(6) step of degrading the RNA of the RNA-DNA double strand using an enzyme having RNase H activity; synthesizing the product [9]
A method for amplifying a target nucleic acid, comprising the following steps (1) to (7).
(1) Using a first primer having a sequence complementary to a portion of the target nucleic acid to which a promoter sequence of an enzyme having RNA polymerase activity is added to the 5′ end side, and an enzyme having DNA-dependent DNA polymerase activity (2) an enzyme having at least strand displacement activity and/or an enzyme having DNA-dependent DNA polymerase activity without 5′→3′ exonuclease activity; (3) a second primer having a sequence homologous to a part of the target DNA and a 3'→5' exonuclease RNA polymerase activity is added to the 5' end of the single-stranded DNA obtained in the step (2) using a DNA-dependent DNA polymerase having no activity and 5'→3' exonuclease activity. (4) from the double-stranded DNA obtained in (3) above, using an enzyme having RNA polymerase activity corresponding to the promoter sequence, step (5) of synthesizing an RNA transcript, cDNA complementary to the target DNA from the RNA transcript synthesized in step (4) above, using a second primer and an enzyme having RNA-dependent DNA polymerase activity; a step of synthesizing
(6) step of degrading the RNA of the RNA-DNA double strand using an enzyme having ribonuclease H (RNase H) activity; synthesizing a transcript [10]
A method for amplifying a target nucleic acid, comprising the following steps (1) to (7).
(1) Using a second primer having a sequence homologous to a portion of the target nucleic acid to which a promoter sequence of an enzyme having RNA polymerase activity is added to the 5′ end side, and an enzyme having DNA-dependent DNA polymerase activity (2) an enzyme having at least strand displacement activity and/or a DNA-dependent DNA polymerase activity having no 5′→3′ exonuclease activity; (3) a first primer having a sequence complementary to a portion of the target DNA, and 3′→5 Using a DNA-dependent DNA polymerase having no 'exonuclease activity and 5'→3' exonuclease activity, from the single-stranded DNA obtained in the step (2), to the 5' end side Step (4) of synthesizing a double-stranded DNA to which a promoter sequence of an enzyme having RNA polymerase activity is added, using an enzyme having RNA polymerase activity corresponding to the promoter sequence, the double strand obtained in (3) above Step (5) of synthesizing an RNA transcript from DNA, using the first primer and an enzyme having RNA-dependent DNA polymerase activity, complementing the target DNA from the RNA transcript synthesized in step (4). a step of synthesizing a specific cDNA;
(6) Step of degrading the RNA of the RNA-DNA double strand using an enzyme having ribonuclease H (RNase H) activity (7) Linking the single-stranded DNA obtained in step (6) to a template synthetically synthesizing an RNA transcript [11]
The step (2) has an enzyme with strand displacement activity and/or an enzyme with DNA-dependent DNA polymerase activity that does not have 5′→3′ exonuclease activity, and the above RNA polymerase activity on the 5′ end side. Using a third primer having a sequence homologous to a portion of the target nucleic acid located at the 5′-end position of the primer to which the promoter sequence of the enzyme was added, the DNA synthesized in step (1) was synthesized. The amplification method according to [9] or [10], which is a step of making the strand DNA.

[12]
(i) an enzyme having RNA-dependent DNA polymerase activity, (ii) an enzyme having DNA-dependent DNA polymerase activity, (iii) an enzyme having RNase H activity, (iv) an enzyme having RNA polymerase activity, (v) a first primer having a sequence complementary to a portion of the target nucleic acid, (vi) a second primer having a sequence homologous to a portion of the target nucleic acid, and (vii) a 3′→5′ exo A first amplification reagent that contains an enzyme that does not have nuclease activity and has DNA-dependent DNA polymerase activity that has 5′→3′ exonuclease activity and does not contain salts (provided that the first primer and the first Either one of the two primers has (iv) a promoter sequence of an enzyme having RNA polymerase activity added to its 5′ end, and a second amplification reagent containing the salt is added to the reaction initiation temperature. temperature control process,
A step of mixing the temperature-controlled amplification reagents so that no reaction occurs;
contacting the mixed reaction liquid with an immiscible immiscible liquid;
agitating immediately before or after contacting said mixed reaction liquid with said immiscible liquid;
A method of amplifying a target nucleic acid, comprising:

[13]
An oligo whose fluorescent properties change compared to before formation when an RNA transcript obtained by the amplification method according to any one of [8] to [12] forms a complementary double strand with a part of the transcript A method for detecting a target nucleic acid using a nucleotide probe.

The present invention will be described in detail below.

<1> Amplification reagent of the present invention The amplification reagent of the present invention is a method capable of amplifying single-stranded RNA at a relatively low constant temperature (for example, 40°C to 50°C) such as the NASBA method, TMA method, or TRC method. It is characterized by adding a DNA-dependent DNA polymerase having no 3'→5' exonuclease activity and having 5'→3' exonuclease activity in addition to the enzymes and primers used. One embodiment of the amplification reagent of the present invention, which is a reagent for amplifying target RNA, is a reagent containing the following components (A) to (I). To either one of (A) and (B) below, a promoter sequence of (G) below is added to the 5'-end side thereof. Also, other components that are more preferred may be added for reasons other than those required for commonly used amplification reactions, while the following is not necessarily exhaustive of the essential reagents for amplification reactions.
(A) a first single-stranded oligonucleotide (first primer) having a sequence complementary to a portion of the target nucleic acid;
(B) a second single-stranded oligonucleotide (second primer) having sequence homology to a portion of the target nucleic acid;
(C) an enzyme having RNA-dependent DNA polymerase activity;
(D) deoxyribonucleoside triphosphates (dNTPs),
(E) an enzyme with RNase H activity;
(F) an enzyme having DNA-dependent DNA polymerase activity;
(G) an enzyme having DNA-dependent RNA polymerase activity;
(H) ribonucleoside triphosphates (NTPs),
(I) a DNA-dependent DNA polymerase without 3′→5′ exonuclease activity and with 5′→3′ exonuclease activity;
The amplified nucleic acid to be amplified with the amplification reagent of the present invention can be arbitrarily determined as long as it contains specific sequence portions in (A) and (B) to the extent that it can be distinguished from other nucleic acids.

As described above, either one of (A) and (B) has the following promoter sequence (G) added to its 5' end. An enhancer sequence consisting of several to several tens of nucleotides may be inserted between the added primer and the promoter sequence.

Of the components (A) to (I), the components (C), (E) and (F) may be different enzymes, or partly or wholly may be a common enzyme. Among them, avian myoblastoma virus (AMV) reverse transcriptase is an enzyme that includes all of the components (C), (E) and (F), and is a particularly preferred embodiment of the amplification reagent of the present invention.

The component (F) is not particularly limited as long as it is an enzyme capable of synthesizing double-stranded DNA from single-stranded DNA, but it is preferable to use an enzyme that does not have 5'→3' exonuclease activity. Examples of the enzyme include 96-7 DNA Polymerase (manufactured by Nippon Gene), DNA Polymerase I Large (Klenow) Fragment, Exonuclease Minus (manufactured by Promega), Bst DNA Polymerase, Large Fragment, and other 3′→5′ exonuclease activities and Enzymes that do not have both 5′→3′ exonuclease activity and 3′→5′ exonuclease activity such as DNA Polymerase I, Large (Klenow) Fragment (manufactured by New England Biolabs) have 5′ → Enzymes without 3′ exonuclease activity can be mentioned. The aforementioned AMV reverse transcriptase is also an enzyme that does not have 5'→3' exonuclease activity.

Examples of components of (G) include T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase. As the promoter sequence to be added to either one of (A) and (B), a sequence corresponding to the component (polymerase) used in (G) may be added.

A feature of the amplification reagent of the present invention is that it contains component (I). Examples of component (I) include Tth DNA polymerase (manufactured by Roche) and Bst DNA Polymerase, Full Length (manufactured by New England Biolabs). In addition, when the component (F) also has the function of (I), it can be considered that the component (I) is included by including the component (F). An example of the component (F) having the function of (I) is HIV reverse transcriptase.

When the primer (A) with the promoter sequence (G) added to the 5′ end side is (A), the amplification of the target RNA by the amplification reagent of this embodiment proceeds in the following steps.
(a-1) (A), (C) and (D) to synthesize cDNA complementary to the target RNA from the target RNA (a-2) (E), the RNA of the RNA-DNA double strand An enzyme having RNA polymerase activity on the 5′-end side of the single-stranded DNA obtained in step (a-2) by steps (a-3) (B), (D) and (I) of degrading step (a-4) of synthesizing a double-stranded DNA to which the promoter sequence of (a-4) is added, by (G) and (H) corresponding to the promoter sequence added in (A), the two strands obtained in (a-3) cDNA complementary to the RNA transcript of (a-4) is synthesized from the strand DNA by the step (a-5) (B), (C) or (I) and (D) of synthesizing the RNA transcript. process,
(a-6) (D), using the single-stranded DNA obtained in the steps (a-7) and (a-6) for degrading the RNA of the RNA-DNA double strand according to (E) as a template; (F), (G) and (H) to synthesize an RNA transcript in a chain reaction. Amplification of target RNA by an amplification reagent proceeds in the following steps.
(b-1) Synthesizing cDNA complementary to the target RNA from the target RNA by (b-1) (A), (C) and (D), by (b-2) (E) RNA of an RNA-DNA double strand An enzyme having RNA polymerase activity on the 5′-end side of the single-stranded DNA obtained in step (b-2) by steps (b-3) (B), (D) and (I) of decomposing Step (b-4) of synthesizing a double-stranded DNA to which the promoter sequence of (B) is added, by (G) and (H) corresponding to the promoter sequence added in (B), the two strands obtained in (b-3) cDNA complementary to the RNA transcript of (b-4) is synthesized from the strand DNA by the step (b-5) (A), (C) or (I) and (D) of synthesizing the RNA transcript. process,
(b-6) by (E), using the single-stranded DNA obtained in the step (b-7) (b-6) of degrading the RNA of the RNA-DNA double strand as a template, (D), (F), (G) and (H) steps of synthesizing RNA transcripts in a concatenated manner When target RNA is amplified using the amplification reagent of the present invention, for example, (A) to (I) described above are used. The reagent may be added to the sample expected to contain the target RNA sequentially according to the steps described above, the components corresponding to the plurality of steps described above may be added together, or all at once may be added.

When the amplification reagent of the present invention is applied to the amplification of target DNA, a method capable of amplifying single-stranded RNA at a relatively low constant temperature (for example, 40°C to 50°C), such as the NASBA method, TMA method, or TRC method. and a DNA-dependent DNA polymerase without 3′→5′ exonuclease activity and with 5′→3′ exonuclease activity, as shown in (J) to (L) below. Either component may be added. However, addition of component (K) below is effective only when (F) is an enzyme such as AMV reverse transcriptase that does not have 5′→3′ exonuclease activity.
(J) Strand Displacement Enzyme (K) Having a sequence homologous to a part of the target nucleic acid located on the 5'-end side of the primer with the promoter sequence of an enzyme having RNA polymerase activity added to the 5'-end side In the case of amplifying the target DNA using the amplification reagent of the present invention to which the components of the third primer (L), (J) and (K) and the component of (K) or (L) are added, 5'→ Since the reaction by an enzyme having no 3' exonuclease activity or a strand displacement enzyme competes with the reaction by (I), it is necessary to adjust the amount of the enzyme so that the former reaction acts predominantly.
The reaction initiation temperature of the present invention is preferably 40°C to 50°C, more preferably 41°C to 46°C.

<2> Micro-compartmentalization method When the amplification reagent of the present invention is used to digitally amplify a target nucleic acid, it is necessary to micro-compartmentize the amplification reagent of the present invention. As a method for microcompartmentalization, a special plate with micropores is used to distribute the reaction liquid into the micropores (micropore distribution method), or a special emulsifier or microchannel chip is used to distribute the reaction liquid. There is a method (droplet method) in which the liquid is divided into a large number of minute droplets (droplets). The size of the microcompartments is preferably 5 nL or less, more preferably 2 nL or less, even more preferably 1 nL or less. QuantStudio 3D digital PCR system manufactured by Thermo Fisher Scientific and Clarity digital PCR system manufactured by JN Medsys are commercially available as apparatuses for microcompartmentalization by the micropore distribution method. As devices for microcompartmentalization by the droplet method, the QX200™ Droplet Digital PCR system manufactured by Bio-Rad and the RainDrop Plus Digital PCR system manufactured by RainDance Technologies are commercially available.

<3> Digital Nucleic Acid Amplification Method When performing digital nucleic acid amplification using the microcompartmentalized reagent in <2>, the two liquids may be mixed to initiate the amplification reaction. For example, there is a method in which droplets are generated (microcompartmentalization) by using a microfluidic chip, immediately after forming a two-liquid parallel flow of the dispersed phase through a Y junction, by the fluid resistance force of the sheath flow of the continuous phase. . The SlipChip is also used to slip two surfaces facing each other (a first surface having a plurality of first microcompartmental areas and a second surface having a plurality of second microcompartmental areas) to slip the plurality of microcompartmental areas. By connecting the first micro-partitioned area and the plurality of second micro-partitioned areas at a ratio of 1:1, the liquids in each micro-partition can be mixed.

Further, a reaction liquid holding part holding two or more reaction liquids, a liquid holding part holding an immiscible liquid immiscible with the reaction liquid, and a laminar flow of the reaction liquids held in each reaction liquid holding part a flow path for contacting the immiscible liquid held by the liquid holding part to form a droplet after the reaction liquid is joined in a state; a droplet holding part for holding the droplet; A microchannel chip having a stirring part provided between a point and an immiscible liquid contact point or between a channel outlet and the droplet holding part, and an outlet provided in the droplet holding part and a temperature control device adjacent to the microchannel chip (for example, the devices shown in FIGS. 2 to 4) may be used for digital nucleic acid amplification. When applying the amplification reagent of the present invention to the reaction apparatus, for example, two or more reaction solutions that do not cause a reaction alone (for example, a first reaction solution containing the components (A) to (I) and not containing salts) and the second reaction liquid containing the salt) are introduced into the reaction liquid holding unit, and after temperature adjustment to the reaction start temperature, the temperature-controlled reaction liquids are combined in a laminar flow state so that the reaction does not occur. (to be in a mixed state), contact with an immiscible liquid to form droplets, and then stir the droplets in a stirring unit (to be in a mixed state) to initiate a nucleic acid amplification reaction. In the present invention, the state of being mixed so as not to cause a reaction is a state in which the state inside the device is expressed in detail. is in a mixed state, but does not react. The reaction is then initiated only after stirring and mixing.

<4> Detection method A target nucleic acid amplified with the amplification reagent of the present invention can be detected by adding a detection component in advance or after the amplification reaction, and measuring the fluorescence or chemiluminescence intensity derived from the component. good. A preferred embodiment of the component for detection is an oligonucleotide probe whose fluorescent properties change when it forms a complementary double strand with a portion of the target nucleic acid compared to before formation.

An example of the probe is a DNA bound with an intercalating fluorescent dye having a sequence complementary or homologous to a portion of the target nucleic acid. The sequence of the DNA portion should be a sequence that is present in the target nucleic acid and that is complementary to a portion that is sufficiently distinguishable from nucleic acids other than the target nucleic acid. The length of the DNA portion is preferably 6 to 100 nucleotides, more preferably 10 to 30 nucleotides, for specific analysis of target nucleic acids. In addition, the 3' end of the DNA portion is non-target nucleic acid so that the 3' end of the DNA portion does not extend from the 3' end by an enzyme having RNA-dependent DNA polymerase activity when complementary bonds are formed with the amplified target nucleic acid. Preferably, a complementary sequence is added or the 3' end is chemically modified (eg aminated).

The intercalating fluorescent dye is such that when the aforementioned DNA portion forms a complementary bond with another nucleic acid, it intercalates with the double-stranded portion and changes its fluorescent properties. For this purpose, for example, an intercalating fluorescent dye may be bound to DNA via a linker of suitable molecular length that does not interfere with intercalation into the double-stranded portion. Such a linker is not particularly limited as long as it is a molecule that does not interfere with the intercalation of the intercalating fluorescent dye into the double-stranded portion. In particular, a linker molecule selected from bifunctional hydrocarbons having functional groups at both ends is convenient and preferred for modifying oligonucleotides. A commercially available reagent set (eg, C6-Thiolmodifier manufactured by Clontech) may also be used.

The intercalating fluorescent dye is not particularly limited as long as it changes its fluorescence characteristics, for example, the wavelength of the emitted fluorescence fluctuates by intercalating into the double strands. From such viewpoints, it is particularly preferable to have a property that fluorescence intensity increases by intercalation. Specifically, thiazole orange, oxazole yellow, and derivatives thereof, which exhibit a particularly remarkable change in fluorescence intensity, can be exemplified as preferred intercalating fluorescent dyes.

The position at which the intercalating fluorescent dye is bound to the DNA portion via the linker is such as the 5′ end, 3′ end, or central portion of the DNA portion, which prevents intercalation of the intercalating fluorescent dye into the double strand. There is no particular limitation as long as it does not inhibit complementary binding between the DNA portion and RNA.

Molecular beacons are another example of oligonucleotide probes that change their fluorescent properties when formed into complementary duplexes with a portion of a target nucleic acid compared to before formation. A molecular beacon is a DNA having a sequence complementary or homologous to a portion of a target nucleic acid, and has a stem-loop structure having a fluorescent dye and a quencher at both ends. In the state of the stem-loop structure, the fluorescence of the fluorescent dye is suppressed by the quencher, but when the region complementary to the target RNA present in the loop sequence hybridizes with the amplification product generated during the reaction, the stem portion is cleaved. and emit light. A stem can be formed by forming a double strand of DNA in a molecule and introducing a pair of a fluorescent dye (such as Cy3) and a quencher (such as an azo compound) with high association ability into the DNA.

Yet another example of an oligonucleotide probe whose fluorescence properties change after forming a complementary double strand with a portion of a target nucleic acid compared to before formation is a FRET (Fluorescence Resonance Energy Transfer) probe. In this case, two probes designed inside the first and second primers used for amplification are used in the sequence of the target nucleic acid. Of the two probes, the 3' end of one probe is modified with a donor fluorescent dye, and the 5' end of the other probe is modified with an acceptor fluorescent dye. By designing the donor fluorescent dye and the acceptor fluorescent dye to be close to each other when the DNA sequences of the two probes are hybridized, the FRET phenomenon occurs when the two probes hybridize with the amplified product generated during the reaction. detectable.

Yet another example of an oligonucleotide probe that undergoes a change in fluorescence properties when formed into a complementary duplex with a portion of a target nucleic acid is a TaqMan probe. In this case, one probe designed inside the first and second primers used for amplification is used in the sequence of the target nucleic acid. The 5' end of the TaqMan probe is labeled with a fluorescent dye (reporter dye), and the 3' end is labeled with a quencher. Although the fluorescence of the reporter dye is suppressed by the quencher, if the probe is hybridized to the target RNA during the extension reaction of the primer by DNA polymerase, the probe is decomposed by the 5′→3′ exonuclease activity and the reporter It can be detected by the fluorescence generated by the liberation of the dye.

The target nucleic acid amplification reagent of the present invention comprises an enzyme having RNA-dependent DNA polymerase activity, an enzyme having DNA-dependent DNA polymerase activity, an enzyme having ribonuclease H (RNase H) activity, and an enzyme having RNA polymerase activity. , a reagent comprising a first primer having a sequence complementary to a portion of the target nucleic acid and a second primer having a sequence homologous to a portion of the target nucleic acid (provided that the first primer and the Either one of the second primers has a promoter sequence of the enzyme having RNA polymerase activity added to its 5' end) and does not have 3'→5' exonuclease activity, and further comprising a DNA-dependent DNA polymerase having 5'→3' exonuclease activity.

INDUSTRIAL APPLICABILITY According to the present invention, amplification efficiency can be dramatically improved in an amplification reaction of a target nucleic acid contained in a sample at a relatively low constant temperature. Therefore, an extremely small amount of target nucleic acid present in a sample can be amplified and detected with higher efficiency and higher reproducibility (higher accuracy) than conventional methods.

FIG. 10 shows the results of Example 3 (fluorescence profile). FIG. 2 is a diagram (plan view) showing a reactor used in Example 5 and Comparative Example 1. FIG. FIG. 3 is a cross-sectional view (front view) of the reactor shown in FIG. 2 taken along line A-A'; FIG. 3 is an enlarged view of a portion surrounded by a dotted square in FIG. 2; 10A and 10B are diagrams showing states of droplets held by a droplet holding unit in Example 5 and Comparative Example 1. FIG.

The present invention will be described in more detail below using Examples and Comparative Examples, but the present invention is not limited to these Examples.

Example 1 Effect of addition of DNA polymerase on RNA detection (part 1)
Effects on RNA detection by further adding various DNA polymerases to a system capable of amplifying single-stranded RNA at a constant temperature (NASBA method, TMA method, TRC method, etc.) were evaluated.

(1) The standard RNA (SEQ ID NO: 1) was prepared by in vitro transcription from a plasmid into which the hepatitis C virus standard RNA (hereinafter also simply referred to as standard RNA) gene was inserted. The standard RNA was diluted with water for injection to 10 ³ copies/2 μL, and this was used as an RNA sample.

(2) After dispensing 10 μL of the reaction solution having the following composition into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), 2 μL of the RNA sample was added. The INAF probe (SEQ ID NO: 2), which is a standard RNA detection probe, binds an intercalating fluorescent dye, oxazole yellow, via a linker between the 10th C and the 11th G of the sequence, It is also an oligonucleotide probe modified with biotin at the 3' end. In addition, the first primer (SEQ ID NO: 3) has a T7 promoter sequence ( It is an oligonucleotide to which SEQ ID NO: 6) is added. The DNA polymerase was any of the DNA polymerases shown in Table 1, and since the concentration (U/μL) of each polymerase was different, the amount added was uniformly 2 μL regardless of the concentration.

Composition of reaction solution: Final concentration after addition of initiator described later (in 20 μL) 66 mM Tris-HCl buffer (pH 8.36)
0.33 mM each dATP, dCTP, dGTP, dTTP
2.0 mM each ATP, CTP, GTP, TTP
3.3mM ITP
122.2 mM trehalose 50 nM INAF probe (portion of homologous strand of standard RNA [107th to 123rd of SEQ ID NO: 1]: SEQ ID NO: 2)
1.0 μM first primer (SEQ ID NO: 3)
1.0 μM second primer (portion of homologous strand of standard RNA [1st to 16th of SEQ ID NO: 1]: SEQ ID NO: 4)
4.3 U AMV reverse transcriptase 95 U T7 RNA polymerase 2 μL additional DNA polymerase or water for injection (3) After incubating the above reaction solution at 46° C. for 3 minutes, 8 μL of an initiator having the following composition was added.

Composition of initiator: Final concentration after addition of initiator (in 20 μL) 18.4 mM magnesium chloride 90.0 mM potassium chloride 0.1% (w/v) Tween 20 (10% Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.) diluted 10 times with water for injection)
9.0% (v/v) DMSO
(4) Subsequently, using a fluorescence spectrophotometer with a temperature control function that can directly measure the PCR tube, react at 46 ° C. and measure the fluorescence intensity (excitation wavelength 470 nm, fluorescence wavelength 520 nm) of the reaction solution over time for 30 minutes. bottom.

Taking the time of the addition of the initiator as 0 minutes, a positive determination was made when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value for a predetermined time by the background fluorescence intensity ratio) exceeded 1.2. A positive judgment was given within 20 minutes as (+), and a positive judgment (negative judgment) was given as (-). Table 1 shows the results.

Among the DNA polymerases examined this time, DNA polymerases with 3'→5' exonuclease activity (DNA Polymerase I and DNA Polymerase I, Large (Klenow) Fragment) are negative regardless of the presence or absence of 5'→3' exonuclease activity. It turned out to be a judgment and greatly inhibit detection. Therefore, it was found that a DNA polymerase having 3'→5' exonuclease activity is not preferable as a DNA polymerase to be added to a system for amplifying single-stranded RNA at a constant temperature.

Example 2 Effect of addition of DNA polymerase on RNA detection (Part 2)
Among the additional DNA polymerases determined positive in Example 1, Bst DNA Polymerase, Full Length, Bst DNA Polymerase, Large Fragment and 96-7 DNA Polymerase were selected and standard RNAs were detected.

(1) After dispensing 10 μL of the reaction solution having the following composition into a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), 2 μL of the RNA sample prepared in Example 1 (1) was added. .

Composition of reaction solution: Final concentration after addition of initiator described later (in 20 μL) 66 mM Tris-HCl buffer (pH 8.36)
0.33 mM each dATP, dCTP, dGTP, dTTP
2.0 mM each ATP, CTP, GTP, TTP
3.3mM ITP
96.3 mM trehalose 50 nM INAF probe (SEQ ID NO:2)
1.0 μM first primer (SEQ ID NO: 3)
1.0 μM second primer (SEQ ID NO: 4)
12.8U AMV Reverse Transcriptase 166U T7 RNA Polymerase Additional DNA Polymerase or Water for Injection (Additional amounts listed in Table 2)
(2) After incubating the above reaction solution at 46° C. for 3 minutes, 8 μL of an initiator having the following composition was added.

Initiator composition: Concentrations are final after addition of initiator (in 20 μL) 18.4 mM Magnesium chloride 90.0 mM Potassium chloride 0.1% (w/v) Tween 20
9.0% (v/v) DMSO
(3) Subsequently, using a fluorescence spectrophotometer with a temperature control function that can directly measure the PCR tube, react at 46 ° C. and measure the fluorescence intensity (excitation wavelength 470 nm, fluorescence wavelength 520 nm) of the reaction solution over time for 30 minutes. bottom.

With the addition of the initiator as 0 minutes, the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value for a predetermined time by the background fluorescence intensity ratio) exceeded 1.2. Table 2 shows the results with time as the detection time.

As a result, 10 U/test is optimal when Bst DNA Polymerase, Full Length is used, 1.6 U/test is optimal when Bst DNA Polymerase, Large Fragment is used, and 8 U/test is optimal for 96-7 DNA Polymerase. was the quantity. The detection time for the RNA sample (standard RNA: 10 ³ copies/test) at the optimum addition amount was 5.5 minutes to 5.7 minutes, and detection in a system without addition of DNA polymerase (system with water for injection added). 0.5 to 0.7 minutes better than the time (6.2 minutes). Generally, in nucleic acid amplification by the TRC method, a 1-minute reduction in detection time is equivalent to a 10-fold increase in sensitivity. I understand.

Example 3 Effect of addition of DNA polymerase on fluorescence intensity ratio Standard RNA was detected in the system in which the amount of additional DNA polymerase added was optimized as determined in Example 2, and fluorescence profiles obtained were compared.

(1) After dispensing 10 μL of the reaction solution having the following composition into a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), 2 μL of the RNA sample prepared in Example 1 (1) was added. .

Composition of reaction solution: Final concentration after addition of initiator described later (in 20 μL) 66 mM Tris-HCl buffer (pH 8.36)
0.33 mM each dATP, dCTP, dGTP, dTTP
2.0 mM each ATP, CTP, GTP, TTP
3.3mM ITP
96.3 mM trehalose 50 nM INAF probe (SEQ ID NO:2)
1.0 μM first primer (SEQ ID NO: 3)
1.0 μM second primer (SEQ ID NO: 4)
12.8U AMV reverse transcriptase 166U T7 RNA polymerase Additional DNA polymerase (either 10U Bst DNA Polymerase, Full Length, 1.6U Bst DNA Polymerase, Large Fragment and 8U 96-7 DNA Polymerase) or water for injection (2) Above After incubating the reaction solution at 46° C. for 3 minutes, 8 μL of an initiator having the following composition was added.

Initiator composition: Concentrations are final after addition of initiator (in 20 μL) 18.4 mM Magnesium chloride 90.0 mM Potassium chloride 0.1% (w/v) Tween 20
9.0% (v/v) DMSO
(3) Subsequently, using a fluorescence spectrophotometer with a temperature control function that can directly measure the PCR tube, react at 46 ° C. and measure the fluorescence intensity (excitation wavelength 470 nm, fluorescence wavelength 520 nm) of the reaction solution over time for 30 minutes. bottom.

FIG. 1 shows a graph (fluorescence profile) plotting the fluorescence intensity ratio of the reaction solution at each measurement time (the value obtained by dividing the fluorescence intensity value at a predetermined time by the background fluorescence intensity ratio) with the time of adding the initiator as 0 minutes. , and the maximum fluorescence intensity ratio are shown in Table 3, respectively. As shown in FIG. 1, after the highest value is measured, the fluorescence intensity ratio almost reaches a plateau, so the maximum value of the fluorescence intensity ratio is considered as the S/N ratio when detecting at an end point or the like. you can

When using a DNA polymerase (Bst DNA Polymerase, Full Length) that does not have 3′→5′ exonuclease activity and has 5′→3′ exonuclease activity among the additional DNA polymerases examined in this example The maximum fluorescence intensity ratio (S/N ratio) was 4.7 times, which is almost equivalent to the system without addition of DNA polymerase (system with water for injection added) (5.0 times). From the above results, systems capable of amplifying single-stranded RNA at a constant temperature (NASBA method, TMA method, TRC method, etc.) do not have 3′→5′ exonuclease activity and 5′→3′ exonuclease activity It can be seen that by further adding a DNA polymerase having nuclease activity, the detection time (sensitivity) can be improved while maintaining the same accuracy (S/N ratio) as before the addition.

On the other hand, the S / N ratio when using a DNA polymerase (Bst DNA Polymerase, Large Fragment and 96-7 DNA Polymerase) that does not have both 3' → 5' exonuclease activity and 5' → 3' exonuclease activity was lower than the system without the addition of DNA polymerase (both 3.4 times).

Example 4 Production of Microchannel Chip In applying the amplification reagent of the present invention to digital nucleic acid amplification, a microchannel chip for digital nucleic acid amplification was produced. Specifically, the microchannel chip 100 shown in FIGS. 2 to 4 was produced using photolithography and soft lithography techniques. The fabrication procedure is shown below.

(1) After dropping a photoresist SU-8 3050 (Microchem) onto a 4-inch bare silicon wafer (Filtech), a photoresist thin film was formed using a spin coater (MIKASA).

(2) Using a mask aligner (Ushio Inc.) and a chromium mask on which the channel pattern of the microchannel chip 100 is formed, the channel pattern is formed on a photoresist film, and then SU-8 Developer (Microchem). was used to develop the channel pattern, to prepare a mold for the channels constituting the microchannel chip 100 (channel height: 80 μm).

(3) In order to suppress adsorption to SU-8, the surface was treated by vapor deposition with Trichloro (1H, 1H, 2H, 2H-perfluoro-octyl) silane (Thermo Fisher Scientific).

(4) A mixture of an uncured siloxane monomer and a polymerization initiator (10:1 weight ratio) prepared using SYLGARD SILICONE ELASTOMER KIT (Dow Corning Toray Co., Ltd.) was applied to the mold treated in (3). By pouring and heating at 80° C. for 2 hours, a polymer (PDMS) substrate 101 on which the shape of the channel was transferred was produced.

(5) The polymer substrate 101 was carefully peeled off from the mold, formed with a cutter, and then formed with a puncher to form an inlet for the reaction liquid 10 and the immiscible liquid 20 and an outlet 80 .

(6) After surface treatment of the polymer substrate 101 with the introduction port and the suction port and the cover glass 102 (Matsunami Glass Co., Ltd.) with an oxygen plasma generator (Meiwa Foursys Co., Ltd.), the patterned surface of the PDMS substrate 101 and the cover glass 102 are attached. Matched.

(7) Ethanol containing 2% Trichloro (1H, 1H, 2H, 2H-perfluoro-octyl) silane (Thermo Fisher Scientific) was introduced into the channel and allowed to stand for 30 minutes to modify the surface of the channel wall surface. , the inside of the channel was washed with ethanol, and air-dried to fabricate the microchannel chip 100 . The prepared chip was stored in a vacuum desiccator.

The fabricated microchannel chip 100 has a size of 24 mm long and 60 mm wide, and has a hole of φ4 mm as an introduction port to the reaction liquid holding parts 10a/10b and the liquid holding part 20, and a hole of φ1.5 mm as an outlet 80. Each hole is provided.

The flow path 11a/11b from the reaction liquid holding section 10a/10b to the reaction liquid confluence section 30 is a meandering flow path with a width of 100 μm and a length of 47 mm. The channels 21a/21b are straight channels having a width of 100 μm and a length of 22 mm and having two bent portions. In the reaction liquid confluence section 30, the reaction liquid flow path 11a and the reaction liquid flow path 11b join at a confluence angle of 90 degrees, and the flow path is narrowed at a rate of 10 degrees from a width of 200 μm to a width of 100 μm. In the droplet forming section 40, the constricted flow path (width 100 μm×length 150 μm) crosses the flow path 21a/21b at an angle of 90 degrees to form a confluence of the reaction liquid and the immiscible liquid. They are brought into contact with each other to form droplets of the reaction liquid that merge in the droplet generation channel (width 80 μm×length 100 μm from the intersection). The droplets formed by the droplet forming section 40 flow to the branching section 60 through the channel outlet 50 (width 200 μm×length 800 μm). The branch flow path 60 has a structure in which it is branched five times in two directions at an angle of 90 degrees. The third branch is 200 μm wide×1.3 mm long, the fourth branch is 200 μm wide×1 mm long, and the fifth branch is 270 μm wide×500 μm long. After being divided into 32 by the branch channel 60, the droplets are introduced into the droplet holding unit 70 (width 15 mm×length 40 mm) and held.

Example 5 Improvement of detection rate in digital nucleic acid amplification by addition of DNA polymerase Applied for digital isothermal nucleic acid amplification of RNA (SEQ ID NO: 1).

(1) HCV standard RNA (SEQ ID NO: 1) was prepared by in vitro transcription from a plasmid into which the HCV gene was inserted. The standard RNA was diluted with water for injection to 10 ⁷ copies/2 μL, and this was used as an RNA sample.

(2) An aqueous solution containing the following composition in 10 μL was prepared and used as a reaction solution containing standard RNA. The molecular beacon probe (SEQ ID NO: 7) is attached to the 5'-end and 3'-end of the homologous strand of the standard RNA, and oligos capable of forming a stem-loop structure when not forming a complementary double strand with the standard RNA. Six nucleotides are added to each (only one base on the 5' side is duplicated), and FAM is bound to the 5' end side, and IowaBlackFQ manufactured by IDT is bound to the 3' end side, respectively. .

132 mM Tris-HCl buffer (pH 8.36)
5.0% (v/v) glycerol 0.66 mM each dATP, dCTP, dGTP, dTTP
4.0 mM each ATP, CTP, GTP, TTP
6.6mM ITP
192.5 mM trehalose 100 nM molecular beacon probe (including part of homologous strand of standard RNA [107 to 123 of SEQ ID NO: 1]: SEQ ID NO: 7)
2.0 μM first primer (SEQ ID NO: 3)
2.0 μM second primer (SEQ ID NO: 4)
2.5 U Bst DNA Polymerase, Full Length
12.8U AMV reverse transcriptase 166U T7 RNA polymerase 10 ⁴ copies standard RNA
(3) An aqueous solution containing the following composition in 10 μL was prepared and used as a starting solution.

36.8 mM magnesium chloride 180.0 mM potassium chloride 0.2% (w/v) Tween 20
18.0% (v/v) DMSO
2.5% (v/v) glycerol 100 nM Molecular Beacon Probe (SEQ ID NO:7)
(4) The microchannel chip 100 produced in Example 4 was fixed on a temperature control block 200 (Mastercycler nexus flat eco, Eppendorf) and heated to 46°C.

(5) After filling the microchannel chip 100 with oil, 100 μL of the oil was dropped onto the liquid holding part 20 .

(6) After removing the oil in the reaction liquid holding parts 10a/10b, the reaction liquid prepared in (2) is applied to the reaction liquid holding part 10a, and the starting solution prepared in (3) is applied to the reaction liquid holding part 10b. 30 μL of each was added dropwise at a liquid temperature of 46°C.

(7) The pump 300 was used to suck the oil from the outlet 80 . One to two minutes after the start of suction, the vicinity of the reaction confluence section 30 became a laminar flow, and the droplet formation section 40 started forming droplets in a ratio of the reaction liquid and the starting liquid at approximately 1:1.

(8) After 3 to 5 minutes from the start of the suction, the suction was stopped and left at 46° C. for 20 minutes to complete the intra-droplet isothermal amplification reaction.

(9) Place the microchannel chip 100 after the reaction in (8) on an inverted microscope IX71 (Olympus), and use a digital CMOS camera (ORCA-FLASH, Hamamatsu Photonics) to capture the droplet holding part. Fluorescence images of droplets held in 70 were acquired.

(10) Using image analysis software (ImageJ), from the fluorescence image, the average diameter of droplets and positive droplets (droplets with a fluorescence intensity ratio of 2.5 times or more with respect to the average fluorescence intensity of negative droplets ) was measured. The average diameter of the droplets was the mean value of the diameters of 9 droplets randomly sampled from the fluorescence image. The number ratio of positive droplets is obtained by first randomly selecting images containing 400 to 700 droplets, measuring the total number of droplets and the number of positive droplets in these images, and adding the measurement results. , the number ratio of positive droplets to a total of 3000 to 6000 droplets was measured.

(11) Using the following (Equation 1) from the average diameter (D [μm]) of the droplets obtained in (10),
After calculating the average diameter of droplets (V [nL]), the number ratio of positive droplets obtained in (10) ( _PP
The standard RNA concentration (C [copy/ _μL ) was calculated using the Poisson distribution approximation (Formula 2) from the average diameter V and the average diameter V, and the detection rate for the added standard RNA (5 × 10 ² copies/μL) was calculated. bottom.

V=(4/3)×π×(D/2) ³ ×10 ⁻⁶ (Formula 1)
C=−ln(1−P _Positive )×(10 ³ /V)×20 (Formula 2)
Comparative example 1
Fluorescent images were obtained in the same manner as in Example 5, except that Bst DNA Polymerase, Full Length was not added in Example 5(2), and the detection rate was calculated.

FIG. 5 shows fluorescence images obtained in Example 5 and Comparative Example 1, and Table 4 shows the results of calculating the detection rate. Adding an enzyme (Bst DNA Polymerase, Full Length) that has DNA-dependent DNA polymerase activity without 3′→5′ exonuclease activity and with 5′→3′ exonuclease activity (Example 5) , it can be seen that the detection rate is improved (Example 5: 62.9%, Comparative Example 1: 54.0%) as compared to when it is not added (Comparative Example 1).

1: Reactor 100: Microchannel chip 101: Polymer substrate 102: Cover glass (glass substrate)
10: reaction liquid holding part 11, 21: channel 20: liquid holding part 30: reaction liquid joining part 40: droplet forming part 50: channel outlet 60: branch channel 70: droplet holding part 80: outlet 200 : Temperature control block 300: Pump

Claims (12)

(i) an enzyme having RNA-dependent DNA polymerase activity;
(ii) an enzyme having DNA-dependent DNA polymerase activity;
(iii) an enzyme having ribonuclease H (RNase H) activity;
(iv) an enzyme having RNA polymerase activity;
(v) a first primer having a sequence complementary to a portion of the target nucleic acid; and (vi) a second primer having a sequence homologous to a portion of the target nucleic acid. , (v) either the first primer or (vi) the second primer has (iv) a promoter sequence for an enzyme having RNA polymerase activity added to its 5′ end, ,
(vii) the amplification reagent further comprising an enzyme having DNA-dependent DNA polymerase activity without 3′→5′ exonuclease activity and with 5′→3′ exonuclease activity. 2. The amplification reagent of claim 1, wherein (ii) the enzyme having DNA-dependent DNA polymerase activity is an enzyme having no 5'->3' exonuclease activity. Claim 1 or 2, wherein (i) the enzyme having RNA-dependent DNA polymerase activity, (ii) the enzyme having DNA-dependent DNA polymerase activity, and (iii) the enzyme having RNase H activity is AMV reverse transcriptase. The amplification reagent described in . 4. The amplification reagent of any one of claims 1-3, further comprising a strand displacement enzyme. A third primer having a sequence homologous to a portion of the target nucleic acid located on the 5'-end side of the target nucleic acid is added to the primer having the promoter sequence of an enzyme having RNA polymerase activity added to the 5'-end side. 5. The amplification reagent of any of claims 1-4, further comprising. (vii) the enzyme having a DNA-dependent DNA polymerase activity that does not have 3′→5′ exonuclease activity and has 5′→3′ exonuclease activity is Bst DNA polymerase (Full Length) or Tth DNA polymerase; 6. The amplification reagent of any one of claims 1-5, wherein the amplification reagent is 7. The target nucleic acid comprising the amplification reagent according to any one of claims 1 to 6 and an oligonucleotide probe whose fluorescence properties change when forming a complementary double strand with a part of the target nucleic acid compared to before formation. Detection reagent. A method for amplifying a target nucleic acid, comprising the following steps (1) to (7) (provided that either the first primer or the second primer has an enzyme having RNA polymerase activity on its 5′ end side) of the promoter sequence is added).
(1) Synthesizing cDNA complementary to the target RNA from the target RNA using a first primer having a sequence complementary to a portion of the target RNA and an enzyme having RNA-dependent DNA polymerase activity (2) ) degrading the RNA of the RNA-DNA duplex using an enzyme having ribonuclease H (RNase H) activity; (3) a second primer having a sequence homologous to a portion of the target RNA; Using a DNA-dependent DNA polymerase that does not have →5′ exonuclease activity and has 5′→3′ exonuclease activity, from the single-stranded DNA obtained in step (2) above, the 5′ end is Step (4) of synthesizing a double-stranded DNA having a promoter sequence of an enzyme having RNA polymerase activity added to the side thereof (4) Using an enzyme having RNA polymerase activity corresponding to the promoter sequence, the double-stranded DNA obtained in (3) is synthesized. Step (5) of synthesizing an RNA transcript from the main strand DNA using a primer having a sequence complementary to a part of the RNA transcript synthesized in the step (4) and an RNA-dependent DNA polymerase activity, the step of synthesizing cDNA complementary to the RNA transcript of (4);
(6) step of degrading the RNA of the RNA-DNA double strand using an enzyme having RNase H activity; Process of synthesizing the product A method for amplifying a target nucleic acid, comprising the following steps (1) to (7).
(1) Using a first primer having a sequence complementary to a portion of the target nucleic acid to which a promoter sequence of an enzyme having RNA polymerase activity is added to the 5′ end side, and an enzyme having DNA-dependent DNA polymerase activity (2) an enzyme having at least strand displacement activity and/or an enzyme having DNA-dependent DNA polymerase activity without 5′→3′ exonuclease activity; (3) a second primer having a sequence homologous to a part of the target DNA and a 3'→5' exonuclease RNA polymerase activity is added to the 5' end of the single-stranded DNA obtained in the step (2) using a DNA-dependent DNA polymerase having no activity and 5'→3' exonuclease activity. (4) from the double-stranded DNA obtained in (3) above, using an enzyme having RNA polymerase activity corresponding to the promoter sequence, step (5) of synthesizing an RNA transcript, cDNA complementary to the target DNA from the RNA transcript synthesized in step (4) above, using a second primer and an enzyme having RNA-dependent DNA polymerase activity; a step of synthesizing
(6) step of degrading the RNA of the RNA-DNA double strand using an enzyme having ribonuclease H (RNase H) activity; Step of synthesizing transcripts A method for amplifying a target nucleic acid, comprising the following steps (1) to (7).
(1) Using a second primer having a sequence homologous to a portion of the target nucleic acid to which a promoter sequence of an enzyme having RNA polymerase activity is added to the 5′ end side, and an enzyme having DNA-dependent DNA polymerase activity (2) an enzyme having at least strand displacement activity and/or a DNA-dependent DNA polymerase activity having no 5′→3′ exonuclease activity; (3) a first primer having a sequence complementary to a portion of the target DNA, and 3′→5 Using a DNA-dependent DNA polymerase having no 'exonuclease activity and 5'→3' exonuclease activity, from the single-stranded DNA obtained in the step (2), to the 5' end side Step (4) of synthesizing a double-stranded DNA to which a promoter sequence of an enzyme having RNA polymerase activity is added, using an enzyme having RNA polymerase activity corresponding to the promoter sequence, the double strand obtained in (3) above Step (5) of synthesizing an RNA transcript from DNA, using the first primer and an enzyme having RNA-dependent DNA polymerase activity, complementing the target DNA from the RNA transcript synthesized in step (4). a step of synthesizing a specific cDNA;
(6) Step of degrading the RNA of the RNA-DNA double strand using an enzyme having ribonuclease H (RNase H) activity (7) Linking the single-stranded DNA obtained in step (6) to a template synthetically synthesizing RNA transcripts The step (2) has an enzyme with strand displacement activity and/or an enzyme with DNA-dependent DNA polymerase activity that does not have 5′→3′ exonuclease activity, and the above RNA polymerase activity on the 5′ end side. Using a third primer having a sequence homologous to a portion of the target nucleic acid located at the 5′-end position of the primer to which the promoter sequence of the enzyme was added, the DNA synthesized in step (1) was synthesized. 11. The amplification method according to claim 9 or 10, which is a step of forming a strand DNA. 12. Oligonucleotide probes whose fluorescent properties change compared to before formation when an RNA transcript obtained by the amplification method according to any one of claims 8 to 11 forms a complementary double strand with a part of the transcript. A method for detecting a target nucleic acid using

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