JP2020162550A - Amplification reagent and amplification method for single-stranded nucleic acid having complementary region at terminal - Google Patents

Abstract

To provide reagents capable of amplifying a single-stranded nucleic acid having a complementary region at least on the 3'-terminal side with high sensitivity rapidly and simply at a constant temperature, without being affected by a double-stranded DNA or AAV vector gene fragments mixed in a sample, and to provide methods for amplifying the single-stranded nucleic acid using the reagent.SOLUTION: Provided is an amplification reagent that contains: an enzyme with an RNA-dependent DNA polymerase activity; an enzyme with a DNA-dependent DNA polymerase activity; an enzyme with a ribonuclease H activity; an enzyme with an RNA polymerase activity; an enzyme with a chain substitution activity; a first primer having a sequence complementary to a part of a region other than a complementary region among single-stranded nucleic acids having the complementary region at least on the 3'-terminal side; and a second primer having a sequence homologous to a part of a region other than the complementary region (however, a promoter sequence of the enzyme with an RNA polymerase activity is added to any one of the first primer and the second primer on its 5'-terminal side). Also provided is an amplification method using the reagent.SELECTED DRAWING: Figure 2

Description

The present invention relates to an amplification method and an amplification reagent for a target nucleic acid contained in a sample. In particular, the present invention relates to an amplification reagent and an amplification method for a single-stranded nucleic acid having a complementary region at the end, such as a viral genome of the Parvoviridae family represented by an adeno-associated virus.

In the treatment of genetic diseases, a method of introducing into a cell using a viral vector containing the gene for the treatment is widely used in terms of high efficiency of introduction into the cell. Among the viral vectors, the adeno-associated virus (AAV) vector is not pathogenic to AAV itself, the gene inserted into the vector can be efficiently introduced into non-dividing cells, and the expression of the introduced gene is sustained for a long period of time. In recent years, it has received particular attention (Non-Patent Document 1).

AAV is a particle with a diameter of 20 nm to 26 nm whose genome is encapsulated in a capsid protein. The genome of AAV is a 4.7 kb linear single-strand DNA, and its 3'end and 5'ends are T-shaped called inverted repeat (hereinafter abbreviated as ITR) sequences. There is a loop structure. When the genome is used as an AAV vector for gene therapy, the Rep gene and Cap gene sandwiched between the ITR sequences on both terminal sides of the genome are used for the promoter sequence and gene therapy specific to the tissue into which the gene is to be introduced. Replace with the gene to be used, poly A signal sequence, etc.

The AAV vector is produced by using an AAV genome replaced with a gene used for gene therapy by the method described above, a vector containing a Rep gene and a Cap gene, and a helper virus such as adenovirus or herpes simplex virus as host cells (HEK293 cells). , HeLa cells, etc.) at the same time to produce. In recent years, instead of the helper virus, a more safe method using an Adv helper plasmid obtained by cloning an adenovirus replication gene has also been performed.

Among the AAV vectors produced by the above-mentioned method, there is also a vector (so-called empty vector) that does not have the AAV genome necessary for gene therapy. Therefore, in order to use the produced AAV vector for gene therapy, it is necessary to confirm whether the AAV genome is present in the vector and to quantify the AAV genome contained in a sample (cell culture solution, etc.) containing the AAV vector. It was necessary.

As qualitative and / or quantitative measurement methods of the AAV genome contained in the AAV vector, image analysis of AAV vector particles by a transmission electron microscope, analytical ultracentrifugal method, quantitative PCR (qPCR) method, and digital droplet PCR (ddPCR) method, dot blot method, and electrophoresis method are known. Of these, the qPCR method is widely used for the quantification of AAV vectors because of its simplicity and speed of operation. However, there are problems such as the fact that the quantification results cannot be correlated with other methods such as the qPCR method and the electrophoresis method, and that the quantification results differ depending on the gene region to be measured (Non-Patent Document 2). 4 and Patent Documents 1 and 2). Further, since the method using PCR such as the qPCR method and the ddPCR method needs to raise and lower the reaction temperature rapidly, there are problems in the labor saving of the reaction process and the cost reduction of the reaction apparatus at the time of automation.

NASBA (Nucleic Acid Sequence) enables nucleic acid amplification at a relatively low temperature (for example, in the range of 40 ° C. to 50 ° C.) as a nucleic acid amplification method that makes it relatively easy to save labor in the reaction process and reduce the cost of the reaction apparatus. Based Amplification
) Method, TMA (Transcription Mediated Amplification) method, and TRC (Transcription Reverse Transcription Concorted) method are known. These amplification methods are usually amplification methods using single-strand RNA as a target nucleic acid, but DNA can also be amplified by devising a reaction system.

For example, Patent Document 3 describes a hepatitis B virus nucleic acid using an enzyme group and a primer set used for RNA amplification by the TRC method, and a reagent containing a strand substitution enzyme and / or a primer for unifying the amplified nucleic acid. Amplification of. Patent Document 4 discloses amplification of hepatitis B virus nucleic acid using a reagent containing an enzyme group and a primer set used for RNA amplification by the TRC method. However, the amplification of single-stranded nucleic acids having complementary regions at the ends, such as the AAV genome, has not been disclosed so far, including Patent Documents 3 and 4.

Japanese Unexamined Patent Publication No. 2003-235562 WO2015 / 080223 JP-A-2015-116136 Japanese Unexamined Patent Publication No. 2014-140367

Takaya Ozawa, Virus, 57 (1), 47-56 (2007) P. Fagone et al. , Human Gene Therapy Methods: B, 23, 1-7 (2012) M. Lock et al. , Human Gene Therapy Methods, 25, 115-125 (2014) S. D'Costa et al. , Methods And Clinical Development 5, 16019 (2016)

The subject of the present invention is that a single-stranded nucleic acid having a complementary region at least on the 3'-terminal side is not affected by the double-stranded DNA or AAV vector gene fragment mixed in the sample, and is highly sensitive at a constant temperature. An object of the present invention is to provide a reagent that can be rapidly and easily amplified and quantified, and a method for amplifying the single-stranded nucleic acid using the reagent.

As a result of diligent studies to solve the above problems, the present inventors have a strand substitution active enzyme and a complementary region at least on the 3'terminal side in the enzyme group used for RNA amplification by the TRC (Transcription Reverse Transcription Concluded) method. Among the single-stranded nucleic acids, the single-stranded nucleic acid is rapidly and highly sensitive at a constant temperature by a reagent to which at least a primer set having a sequence complementary and homologous to a part of a region other than the complementary region is added. We have found that amplification and quantification can be performed easily, and have completed the present invention.

That is, the first aspect of the present invention is
It is an amplification reagent for a single-stranded nucleic acid having at least a complementary region on the 3'-terminal side, which contains at least the following (1) to (7) (however, one of the following (6) and (7). The promoter sequence of the enzyme of (4) below is added to the 5'terminal side thereof).
(1) An enzyme having RNA-dependent DNA polymerase activity;
(2) Enzyme having DNA-dependent DNA polymerase activity;
(3) Enzyme having ribonuclease H activity;
(4) Enzyme having RNA polymerase activity;
(5) Enzyme having chain substitution activity;
(6) A first primer having a sequence complementary to a part of a region other than the complementary region of the single-stranded nucleic acid; and (7) A region of the single-stranded nucleic acid other than the complementary region. A second primer having a sequence homologous to a portion of.

The second aspect of the present invention is the amplification reagent according to the first aspect, wherein the (1), (2) and (3) are AMV reverse transcriptases.

Furthermore, the third aspect of the present invention is
A method for amplifying a single-stranded nucleic acid having a complementary region at least on the 3'-terminal side, which comprises at least the following steps (I) to (VIII).
(I) Among the single-stranded nucleic acids, a first primer having a sequence complementary to a part of a region other than the complementary region, an enzyme having DNA-dependent DNA polymerase activity, and an enzyme having strand substitution activity. To synthesize DNA complementary to the single-stranded nucleic acid using
(II) A step of converting the DNA synthesized in (I) into a single-stranded DNA having a complementary region on the 3'end side using an enzyme having a strand substitution activity;
(III) Of the single-stranded nucleic acids, a second primer having a sequence homologous to a part of a region other than the complementary region, an enzyme having DNA-dependent DNA polymerase activity, and an enzyme having strand substitution activity. A step of synthesizing DNA homologous to the single-stranded nucleic acid using and.
(IV) A promoter of an enzyme having RNA polymerase activity possessed by either one of the first primer and the second primer by using the first primer and an enzyme having DNA-dependent DNA polymerase activity. A step of synthesizing double-stranded DNA from the DNA synthesized in (III) above with a sequence added;
(V) A step of synthesizing an RNA transcript from the double-stranded DNA using an enzyme having RNA polymerase activity corresponding to the promoter sequence;
(VI) Complementary to the RNA transcript using a primer having a sequence complementary to the RNA transcript among the first primer and the second primer and an enzyme having RNA-dependent DNA polymerase activity. Steps of synthesizing typical cDNA;
(VII) A step of converting the cDNA into a single-stranded DNA using an enzyme having ribonuclease activity; and (VIII) using the single-stranded DNA obtained in the above (VII) as a template, continuously producing RNA transcripts. The process of synthesizing.
In addition, each of the above steps may be performed simultaneously, continuously and / or automatically.

A fourth aspect of the present invention is a step of synthesizing an RNA transcript using the method for amplifying a single-stranded nucleic acid according to the third aspect; and forming a double strand complementary to a part of the RNA transcript. This is a method for detecting a single-stranded nucleic acid having a complementary region at least on the 3'-terminal side, which includes a step of detecting an RNA transcript using an oligonucleotide probe whose fluorescence characteristics change as compared with that before formation. ..

Furthermore, the fifth aspect of the present invention is a method for evaluating the quality of a sample containing a viral vector, and the single-stranded nucleic acid in the method for detecting a single-stranded nucleic acid according to the fourth aspect is one of the viral vectors. A sample containing the viral vector, which is a full-stranded nucleic acid, is detected qualitatively and / or quantitatively by using the method for detecting the single-stranded nucleic acid and contained in the sample, and based on the detection result. It is a method including a step of evaluating the quality of the nucleic acid.

The amplification reagent of the present invention has an enzyme having RNA-dependent DNA polymerase activity, an enzyme having DNA-dependent DNA polymerase activity, an enzyme having ribonuclease H activity, an enzyme having RNA polymerase activity, and a strand substitution activity. The enzyme and the first primer having a sequence complementary to a part of the region other than the complementary region among the single-stranded nucleic acids having a complementary region at least on the 3'end side, and the region other than the complementary region. A second primer having a sequence homologous to a part of (however, one of the first primer and the second primer has the enzyme polymerase activity on the 5'end side thereof. ), And is characterized by including.

With the amplification reagent of the present invention, the single-stranded nucleic acid can be rapidly amplified with high specificity, high sensitivity, and a method of amplifying nucleic acid at a constant temperature such as NASBA method, TMA method, and TRC method. When the single-stranded nucleic acid amplified by the amplification reagent of the present invention contains a complementary region on the 5'terminal side, setting an oligonucleotide probe for detection in the complementary region causes a problem in detection by the PCR method. The problem that the inserted target gene is detected at the same time can be solved.

It is a figure which showed the region (base number 4526-4675 of SEQ ID NO: 20) containing the inverted repeat (ITR) sequence on the 3'-terminal side in the adeno-associated virus serotype 2 (AAV2) genome. Base numbers 4551-4675 of SEQ ID NO: 20 correspond to the ITR sequence. Amplification of the present invention when the single-stranded nucleic acid to be amplified is a single-stranded DNA having complementary regions on the 5'end side and the 3'end side and the other regions do not have complementary regions. It is a figure which showed an example of the amplification method of a single-stranded nucleic acid using a reagent. Amplification of the present invention when the single-stranded nucleic acid to be amplified is a single-stranded DNA having complementary regions on the 5'end side and the 3'end side and the other regions do not have complementary regions. It is a figure which showed another example of the amplification method of a single-stranded nucleic acid using a reagent. The present invention is a case where the single-stranded nucleic acid to be amplified is a single-stranded DNA having complementary regions on the 5'-terminal side and the 3'-terminal side, and also having complementary regions on other regions. It is a figure which showed still another example of the amplification method of the single-stranded nucleic acid using the amplification reagent of. The present invention is a case where the single-stranded nucleic acid to be amplified is a single-stranded DNA having complementary regions on the 5'-terminal side and the 3'-terminal side, and also having complementary regions on other regions. It is a figure which showed still another example of the amplification method of the single-stranded nucleic acid using the amplification reagent of. When the single-stranded nucleic acid to be amplified is a single-stranded DNA having a complementary region on the 3'end side and the other regions do not have a complementary region, the amplification reagent of the present invention is used. It is a figure which showed an example of the amplification method of a double-stranded nucleic acid.

Hereinafter, the present invention will be described in detail.
One aspect of the present invention is an amplification reagent for a single-stranded nucleic acid having a complementary region at least on the 3'-terminal side, which comprises at least the following (1) to (7) (however, the following (6) and (7)). One of them is related to (the promoter sequence of the enzyme of (4) below is added to the 5'terminal side thereof).
(1) An enzyme having RNA-dependent DNA polymerase activity;
(2) Enzyme having DNA-dependent DNA polymerase activity;
(3) Enzyme having ribonuclease H activity;
(4) Enzyme having RNA polymerase activity;
(5) Enzyme having chain substitution activity;
(6) A first primer having a sequence complementary to a part of a region other than the complementary region of the single-stranded nucleic acid; and (7) A region of the single-stranded nucleic acid other than the complementary region. A second primer having a sequence homologous to a portion of.

<Single-stranded nucleic acid>
The single-stranded nucleic acid to be amplified by the present invention is a single-stranded nucleic acid region amplified by the amplification reagent and amplification method of the present invention, or a nucleic acid including a complementary region having at least 3'-terminal side including the region. It means the whole thing. The single-stranded nucleic acid is preferably DNA.

The single-stranded nucleic acid amplification reagent of the present invention can be applied to any single-stranded nucleic acid having a complementary region at least on the 3'terminal side. The single-stranded nucleic acid may further have a complementary region on the 5'terminal side. When DNA is targeted for amplification, artificially prepared DNA, environmental samples, and DNA existing in living organisms are exemplified. Although the present invention is not particularly limited, a virus, preferably AAV, is exemplified as the origin of DNA.

In the present specification, the complementary region is a nucleic acid inside the single-stranded nucleic acid (5'-terminal complementary region) via a loop structure in which several bases to several tens of bases of the single-stranded nucleic acid are composed of several bases to several tens of bases. In the case of a nucleic acid located on the 3'-terminal side via a loop structure, in the case of a 3'-terminal complementary region, a nucleic acid located on the 5'-terminal side via a loop structure) hybridizes under stringent conditions and doubles. A region in which a chain is formed (double chain formation region). The single-stranded nucleic acid to be amplified by the present invention has a complementary region at least on the 3'terminal side. The base at the end of the complementary region hybridizes with the corresponding base. However, among single-stranded nucleic acids, the 5'terminal region of the complementary region on the 3'end side (when the single-stranded nucleic acid has complementary regions on both the 5'end side and the 3'end side). Is a region sandwiched between complementary regions on both terminal sides), and the complementary double strand derived from the secondary structure of the single-stranded nucleic acid is included in the complementary region used in the amplification step of the present invention. Absent.

Here, the stringent condition means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. To give an example, polynucleotides having high homology (for example, identity or similarity) have homology of, for example, 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more. It is a condition that the polynucleotides having them hybridize with each other and the polynucleotides showing lower homology do not hybridize with each other. Specifically, as an example of stringent conditions in the present invention, at 42 ° C., 50% (v / v) formamide, 0.1% (w / v) bovine serum albumin, 0.1% (w). Conditions include the presence of / v) ficol, 0.1% (w / v) polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 150 mM sodium chloride, and 75 mM sodium citrate.

Of the complementary regions, the length of the double chain forming region is not particularly limited as long as it can form a double chain under the above stringent conditions while maintaining the state via the loop structure. As an example, it is 2 to 150 bases, 2 to 100 bases, 2 to 50 bases, or 2 to 30 bases.

The loop structure in the complementary region means a structure in which at least one base of the region sandwiched between the double-stranded nucleic acid-forming regions does not hybridize with the nucleic acid inside the single-stranded nucleic acid. However, a part of the loop structure may have a region forming a double strand with the nucleic acid inside the single-stranded nucleic acid. The base length forming the loop structure is not particularly limited, and for example, it is 1 to 100 bases, 1 base to 50 bases, 1 base to 40 bases, or 1 base to 30 bases. The base sequence of the loop structure may be any sequence as long as it does not interfere with hybridization in the duplex formation region in the complementary region.

An example of a loop structure having a region that forms a double strand with a nucleic acid inside a single-stranded nucleic acid is an inverted repeat (ITR) sequence of an adeno-associated virus serotype 2 (AAV2) genome (Fig.). 1). In FIG. 1, the complementary region in the present invention is the region of ABCB'-D-E-F-E'-A', and the duplex forming region is the region of AA'. The loop structure refers to the region of B-C-B'-D-E-FE', respectively.

The amount of the single-stranded nucleic acid added to the reaction system can be appropriately set according to the required amount of the target substance and the like with reference to the description in the present specification and known nucleic acid amplification techniques.

<Primer>
The primer used in the amplification reagent of the present invention is
Among the single-stranded nucleic acids to be amplified, the first primer having a sequence complementary to a part of the region other than the above-mentioned complementary region, and among the single-stranded nucleic acids to be amplified, other than the above-mentioned complementary region A second primer with a sequence homologous to part of the region,
However, a promoter sequence of an enzyme having RNA polymerase activity, which will be described later, is added to either one of the first primer and the second primer on the 5'terminal side thereof.

In the present specification, "having a complementary sequence" means having a sequence capable of hybridizing to the target nucleic acid under stringent conditions, and "having a homologous sequence" means complementing the target nucleic acid. It means having a sequence capable of hybridizing to a strand under stringent conditions. Therefore, as long as the hybridization is maintained, the primer may be substituted, deleted, added, or modified by about 1 or several bases (for example, about 10% or less of the primer sequence). The length of the primer can be set arbitrarily, but is preferably in the range of 10 to 50 bases. Specific examples of the primer include, but are not limited to, those having the sequences shown in SEQ ID NOs: 11 to 17.
Primers can be used alone or in combination of two or more. The amount of the primer added to the reaction system can be appropriately determined according to the reaction system with reference to the description in the present specification and known nucleic acid amplification techniques. Primers can be designed and manufactured based on the description and known techniques herein.

For example, in the case of a single-stranded nucleic acid having an AAV2 genome ITR (FIG. 1) on the 5'end side and the 3'end side, it has a sequence complementary to a part of the region other than the ITR and has a sequence complementary to a part of the region other than the ITR. As the first primer used in the amplification reagent of the present invention, an oligonucleotide in which a promoter sequence of an enzyme having RNA polymerase activity described later is added to the 5'end side of the sequence is used as a region other than the ITR (however, the first Oligonucleotides having a sequence homologous to a part of (position on the 3'-terminal side of the single-stranded nucleic acid) with respect to the primer may be used as the second primer used in the amplification reagent of the present invention. In the above-mentioned example, the promoter sequence of the enzyme having RNA polymerase activity is added to the 5'end side of the first primer, but the promoter sequence may be added to the 5'end side of the second primer. Good (in which case it is not necessary to add the promoter sequence to the first primer). Further, it is preferable that the complementary sequence is set at a position 3'-terminal side from the central portion of the single-stranded nucleic acid.

<Enzyme with RNA polymerase activity>
Examples of the enzyme having RNA polymerase activity used in the amplification reagent of the present invention include, but are not limited to, bacteriophage-derived T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, or derivatives thereof, which are widely used in molecular biological experiments and the like. As the promoter sequence of the enzyme having RNA polymerase activity added to the above-mentioned primer, a sequence corresponding to the enzyme having RNA polymerase activity used in the present invention may be used. When T7 RNA polymerase is used as an enzyme having RNA polymerase activity, for example, a known T7 promoter may be used. One or a combination of two or more of these can be used. The amount of the enzyme having RNA polymerase activity added to the reaction system may be appropriately determined according to the reaction system with reference to the description in the present specification and known nucleic acid amplification techniques.

<Enzyme with RNA-dependent DNA polymerase activity, enzyme with DNA-dependent DNA polymerase activity, enzyme with ribonuclease H activity>
In addition to the above-mentioned enzyme having RNA polymerase activity, the amplification reagent of the present invention includes an enzyme having at least RNA-dependent DNA polymerase activity, an enzyme having DNA-dependent DNA polymerase activity, and ribonuclease H (RNase H) activity. It also contains the enzymes it has. As the enzyme having RNA-dependent DNA polymerase activity, the enzyme having DNA-dependent DNA polymerase activity, and the enzyme having RNaseH activity, an enzyme having several activities may be used, and a plurality of enzymes having each activity may be used. You may use it. Tori myeloblastopathy virus (AMV) reverse transcriptase, murine leukemia virus (MMLV) reverse transcriptase, human immunodeficiency virus (HIV) reverse transcriptase and their derivatives have all of the three enzyme activities described above. Therefore, AMV reverse transcriptase and its derivatives are particularly preferable. One or a combination of two or more of these can be used. The amount of each enzyme added to the reaction system may be appropriately determined according to the reaction system with reference to the description in the present specification and known nucleic acid amplification techniques.

<Enzyme with chain substitution activity>
The enzyme having a strand substitution activity used in the amplification reagent of the present invention refers to an enzyme that synthesizes a new DNA strand while dissociating the hydrogen bond of the double-stranded DNA as a template by itself. If the enzyme having DNA-dependent DNA polymerase activity also functions as an enzyme having strand substitution activity, an enzyme having these activities can be used as a DNA-dependent DNA polymerase and an enzyme having strand substitution activity. You may use it. Specifically, but not limited to, E.I. colli DNA polymerase I, Klenow fragment of DNA polymerase I, T7 or T5 bacteriophage DNA polymerase, trimyeloblastosis virus (AMV) reverse transcriptase, mouse leukemia virus (MMLV) reverse transcriptase, human immunodeficiency virus (HIV) ) Reverse transcriptase, Bsu DNA polymerase, Bst DNA polymerase, Aac DNA polymerase, phi29 DNA polymerase, 96-7 DNA polymerase, Bca DNA polymerase, and derivatives thereof. Helicase can also be used as an enzyme having a strand substitution activity in the present invention because it produces a strand substitution effect, that is, a nucleic acid substitution associated with nucleic acid synthesis of the same sequence. Further, RecA and a single-strand-binding protein can also be used as an enzyme having a chain-substituting activity in the present invention. One or a combination of two or more of these can be used. The amount of the enzyme having a chain substitution activity added to the reaction system may be appropriately determined according to the reaction system with reference to the description in the present specification and known nucleic acid amplification techniques.

<Other ingredients>
In addition to the above components, the amplification reagents of the present invention include components used for any reaction, detection, etc., such as reaction buffers, divalent metal ions, deoxyribonucleotides, and oligos, as long as the effects of the present invention are not impaired. It may contain at least one selected from nucleotide probes, intercalating dyes and the like.

A sample containing a single-stranded nucleic acid having complementary regions on the 5'end side and the 3'end side, which are target nucleic acids, is added to the amplification reagent of the present invention, and the single-stranded nucleic acid is reacted at a constant temperature. Nucleic acid (more precisely, RNA transcript) can be amplified. That is, the amplification reagent of the present invention can be suitably applied to methods for amplifying nucleic acids at a constant temperature, such as the NASBA method, the TMA method, and the TRC method. The reaction temperature depends on the heat resistance and activity of each enzyme used, the Tm of the primer, etc., but the enzymes used are AMV reverse transcriptase and T7 RNA polymerase, and the primer length is 17 to 30 bases. In the case of a range, the reaction temperature may be set in the range of 35 ° C. to 65 ° C., and more preferably set in the range of 40 ° C. to 50 ° C. The reaction time can be appropriately set according to the required amount of the target substance and the like.

<Detection method>
By detecting the single-stranded nucleic acid (RNA transcript) amplified by the amplification reagent and the amplification method of the present invention (performing the detection method of the present invention), the presence or absence of the single-stranded nucleic acid in the measurement sample and the presence or absence of the single-stranded nucleic acid in the sample can be detected. The amount of single-stranded nucleic acid contained can be measured. A conventionally known nucleic acid detection method can be used for detection. In particular,
(I) Method using electrophoresis or liquid chromatography,
(Ii) Hybridization method using an oligonucleotide probe labeled with a detectable label,
(Iii) A method using a fluorescent dye-labeled oligonucleotide probe designed so that the fluorescence characteristics are changed by hybridizing (forming a complementary double strand) with a part of the base sequence of the single-stranded nucleic acid.
And so on. As the labeled oligonucleotide probe, those synthesized based on the description described in the present specification and known synthetic techniques, or commercially available ones can be used. Examples of the fluorescent dye-labeled probe of (iii) include a fluorescent-labeled probe using FRET (fluorescence resonance energy transfer) and an oligonucleotide probe labeled with an intercalating fluorescent dye.

As an example of the oligonucleotide probe labeled with the intercalating fluorescent dye, an oligonucleotide capable of specifically hybridizing with a part of nucleic acid containing a specific base sequence of amplified single-stranded nucleic acid under stringent conditions. There are oligonucleotide probes labeled with an intercalator fluorescent dye via a suitable linker on the 3'end side, 5'end side, phosphate diester portion or base moiety. When the probe forms a double strand complementary to the specific base sequence of the amplified single-stranded nucleic acid, the intercalating fluorescent dye portion intercalates into the complementary double-stranded portion to change the fluorescence characteristics. Is. The intercalator fluorescent dye to be labeled is not particularly limited, and among general-purpose fluorescent dyes such as oxazole yellow, thiazole orange, ethidium bromide, and hemicianin, and derivatives thereof, the fluorescence intensity and fluorescence characteristics are taken into consideration. It may be selected as appropriate. Except when the fluorescent dye is labeled on the 3'-terminal side, the 3'-terminal side of the oligonucleotide may be appropriately modified with glycolic acid or the like in order to prevent a nucleic acid extension reaction from the terminal side. ..

A preferred example of the oligonucleotide constituting the oligonucleotide probe labeled with the intercalating fluorescent dye is an oligonucleotide capable of specifically hybridizing with the amplified single-stranded nucleic acid under stringent conditions. When such an oligonucleotide probe is added to the amplification reagent of the present invention in advance and the change in fluorescence characteristics is measured over time with a fluorescence spectrophotometer, the amplification and detection of the target nucleic acid contained in the sample is sealed in one step. It is preferable because it can be performed in a container. Further, since the fluorescence intensity is measured over time, the measurement can be completed at any time when a significant increase in fluorescence is observed, and the nucleic acid amplification and the measurement can be completed within 20 minutes in general.

According to the detection method of the present invention, the quality of a vector having a single-stranded nucleic acid having a complementary region at least on the 3'end side can be evaluated. As a specific example, at least the 3'end side is a complementary region of a viral genome such as ITR (FIG. 1) of the AAV genome, and the target gene is inserted into the 5'end side region of the complementary region having the 3'end side. , When evaluating a viral vector, a primer may be designed at the position of the target gene and the detection method of the present invention may be performed. If it can be detected, it can be seen that the target gene has been inserted into the viral vector, and if it cannot be detected, it can be seen that the target gene has not been inserted into the viral vector (so-called empty vector) (qualitative). Detection). If it can be detected, it can be evaluated whether the amount of viral vector contained in the sample is appropriate by measuring the amount (quantitative detection).

Hereinafter, the present invention will be described in more detail with reference to the drawings, but the present invention is not limited to the embodiments described in these figures.

The single-stranded nucleic acid to be amplified has a complementary region on the 5'end side and the 3'end side, and the other region (the region sandwiched between the two complementary regions) does not have a complementary region. The flow of single-stranded nucleic acid amplification using the amplification reagent of the present invention when the primer to which the promoter sequence of the enzyme having RNA polymerase activity is added is the second primer will be described below (FIG. 2). ). Since the following steps (A-10) and subsequent steps are the same as the amplification steps by the usual TRC (Transcription Reverse-transcription Concerted) method, they are abbreviated as "TRC reaction" in FIG.

(A-1) Of the single-stranded DNA (template single-stranded DNA) to be amplified, the first primer having a sequence complementary to a part of the region other than the complementary region is used as the single-stranded DNA. (Addition of antisense primer).
(A-2) The single-stranded DNA derived from the first primer and the template single-stranded DNA are extended by an enzyme having DNA-dependent DNA polymerase activity and an enzyme having strand substitution activity.
(A-3) The single-stranded DNA derived from the first primer is released from the template single-stranded DNA by an enzyme having strand substitution activity and an elongated template single-stranded DNA.
(A-4) It has a sequence complementary to a part of the single-stranded DNA derived from the first primer (homogeneous to the template single-stranded DNA), and is located on the 5'end side of the sequence. The second primer to which the promoter sequence of the enzyme having RNA polymerase activity is added is hybridized to the single-stranded DNA derived from the first primer released in (A-3) (addition of sense primer). (A-5) A single-stranded DNA derived from the first primer and a single-stranded DNA to which a promoter is added (derived from the second primer) are extended by an enzyme having DNA-dependent DNA polymerase activity.
(A-6) The promoter-added single-stranded DNA is released from the single-stranded DNA derived from the first primer by an enzyme having strand substitution activity and a single-stranded DNA derived from the extended first primer.
(A-7) The first primer is hybridized to the single-stranded DNA to which the promoter released in (A-6) is added (addition of antisense primer).
(A-8) A double-stranded DNA having a promoter sequence added to the 5'end side is synthesized by an enzyme having DNA-dependent DNA polymerase activity.
(A-9) An RNA polymerase corresponding to the promoter is allowed to act on the promoter-added double-stranded DNA synthesized in (A-8) to synthesize an RNA transcript (amplification of the RNA transcript).
(A-10) The first primer is hybridized to the RNA transcript (addition of antisense primer).
(A-11) Synthesis of cDNA complementary to RNA transcript is performed by an enzyme having RNA-dependent DNA polymerase activity (synthesis of RNA-DNA double strand).
(A-12) The cDNA synthesized in (A-11) by an enzyme having ribonuclease H (RNase H) activity is converted into single-stranded DNA (degradation of RNA transcript).
RNA transcripts are continuously synthesized using the single-stranded DNA obtained in (A-13) and (A-12) as a template (amplification of RNA transcripts).

Next, the single-stranded nucleic acid to be amplified has complementary regions on the 5'end side and 3'end side, and the other regions (regions sandwiched between both complementary regions) do not have complementary regions. The flow of single-stranded nucleic acid amplification using the amplification reagent of the present invention when the primer which is single-stranded DNA and to which the promoter sequence of the enzyme having RNA polymerase activity is added is the first primer will be described below. (Fig. 3). In FIG. 3, as in FIG. 2, the amplification step by the TRC method (the following steps after (B-10)) is abbreviated as “TRC reaction”.

(B-1) Of the single-stranded DNA to be amplified (primer single-stranded DNA), it has a sequence complementary to a part of a region other than the complementary region, and RNA is located on the 5'end side of the sequence. The first primer to which the promoter sequence of the enzyme having polymerase activity is added is hybridized to the single-stranded DNA (addition of antisense primer).
(B-2) The single-stranded DNA derived from the first primer and the template single-stranded DNA are extended by an enzyme having DNA-dependent DNA polymerase activity and an enzyme having strand substitution activity.
(B-3) The single-stranded DNA derived from the first primer is released from the template single-stranded DNA by an enzyme having strand substitution activity and an elongated template single-stranded DNA.
(B-4) A second primer having a sequence complementary to a part of the single-stranded DNA derived from the first primer (homologous to the template single-stranded DNA) was added to (B-3). ) Is hybridized with the single-stranded DNA derived from the first primer (addition of sense primer).
(B-5) A single-stranded DNA derived from the first primer and a single-stranded DNA derived from the second primer are extended by an enzyme having DNA-dependent DNA polymerase activity.
(B-6) The single-stranded DNA derived from the second primer is released from the single-stranded DNA derived from the first primer by the enzyme having the strand substitution activity and the single-stranded DNA derived from the extended first primer. ..
(B-7) The first primer is hybridized to the single-stranded DNA derived from the second primer released in (B-6) (addition of antisense primer).
(B-8) A double-stranded DNA having a promoter sequence added to the 5'end side is synthesized by an enzyme having DNA-dependent DNA polymerase activity.
An RNA polymerase corresponding to the promoter is allowed to act on the promoter-added double-stranded DNA synthesized in (B-9) and (B-8) to synthesize an RNA transcript (amplification of the RNA transcript).
(B-10) The second primer is hybridized to the RNA transcript (addition of sense primer).
(B-11) Synthesis of cDNA complementary to RNA transcript is performed by an enzyme having RNA-dependent DNA polymerase activity (synthesis of RNA-DNA double strand).
(B-12) The cDNA synthesized in (B-11) by an enzyme having ribonuclease H (RNase H) activity is converted into single-stranded DNA (degradation of RNA transcript).
RNA transcripts are continuously synthesized using the single-stranded DNA obtained in (B-13) and (B-12) as a template (amplification of RNA transcripts).

Next, the single-stranded nucleic acid to be amplified has complementary regions on the 5'end side and 3'end side, and also has complementary regions in other regions (regions sandwiched between both complementary regions). The flow of single-stranded nucleic acid amplification using the amplification reagent of the present invention when the primer to which the promoter sequence of the enzyme having RNA polymerase activity is added is the second primer is as follows. This will be described (Fig. 4). In FIG. 4, as in FIG. 2, the amplification step by the TRC method (the following steps after (C-11)) is abbreviated as “TRC reaction”.

(C-1) The single-stranded DNA (template single-stranded DNA) to be amplified is extended by an enzyme having DNA-dependent DNA polymerase activity and an enzyme having strand substitution activity, and the binding in the complementary region is dissociated.
(C-2) The first primer having a sequence complementary to a part of the region dissociated in (C-1) is hybridized to the single-stranded DNA (addition of antisense primer).
(C-3) A single-stranded DNA derived from the first primer and a template single-stranded DNA are extended by using an enzyme having DNA-dependent DNA polymerase activity and an enzyme having strand substitution activity.
(C-4) The single-stranded DNA derived from the first primer is released from the template single-stranded DNA by an enzyme having strand substitution activity and an elongated template single-stranded DNA.
(C-5) Has a sequence complementary to a part of the single-stranded DNA derived from the first primer (homogeneous to the template single-stranded DNA), and is located on the 5'end side of the sequence. The second primer to which the promoter sequence of the enzyme having RNA polymerase activity is added is hybridized to the single-stranded DNA derived from the first primer released in (C-4) (addition of sense primer). (C-6) A single-stranded DNA derived from the first primer and a single-stranded DNA (derived from the second primer) to which a promoter is added are extended by an enzyme having DNA-dependent DNA polymerase activity.
(C-7) The single-stranded DNA to which the primer is added is released from the single-stranded DNA derived from the first primer by the enzyme having the strand substitution activity and the single-stranded DNA derived from the extended first primer.
(C-8) The first primer is hybridized to the single-stranded DNA to which the primer released in (C-7) is added (addition of antisense primer).
(C-9) A double-stranded DNA having a promoter sequence added to the 5'end side is synthesized by an enzyme having DNA-dependent DNA polymerase activity.
An RNA polymerase corresponding to the promoter is allowed to act on the promoter-added double-stranded DNA synthesized in (C-10) and (C-9) to synthesize an RNA transcript (amplification of the RNA transcript).
(C-11) The first primer is hybridized to the RNA transcript (addition of antisense primer).
(C-12) Synthesis of cDNA complementary to RNA transcript is performed by an enzyme having RNA-dependent DNA polymerase activity (synthesis of RNA-DNA double strand).
(C-13) The cDNA synthesized in (C-12) by an enzyme having ribonuclease H (RNase H) activity is converted into single-stranded DNA (degradation of RNA transcript).
RNA transcripts are continuously synthesized using the single-stranded DNA obtained in (C-14) and (C-13) as a template (amplification of RNA transcripts).

Next, the single-stranded nucleic acid to be amplified has complementary regions on the 5'end side and 3'end side, and also has complementary regions in other regions (regions sandwiched between both complementary regions). The flow of single-stranded nucleic acid amplification using the amplification reagent of the present invention when the primer to which the promoter sequence of the enzyme having RNA polymerase activity is added is the first primer is as follows. This will be described (Fig. 5). In FIG. 5, as in FIG. 2, the amplification step by the TRC method (the following steps after (D-11)) is abbreviated as “TRC reaction”.

(D-1) The single-stranded DNA (template single-stranded DNA) to be amplified is extended by an enzyme having DNA-dependent DNA polymerase activity and an enzyme having strand substitution activity, and the binding in the complementary region is dissociated.
(D-2) A first sequence having a sequence complementary to a part of the region dissociated in (D-1) and having a promoter sequence of an enzyme having RNA polymerase activity added to the 5'terminal side of the sequence. The primer is hybridized to the single-stranded DNA (addition of antisense primer). (D-3) A single-stranded DNA derived from the first primer and a template single-stranded DNA are extended by using an enzyme having DNA-dependent DNA polymerase activity and an enzyme having strand substitution activity.
(D-4) The single-stranded DNA derived from the first primer is released from the template single-stranded DNA by an enzyme having strand substitution activity and an elongated template single-stranded DNA.
(D-5) A second primer having a sequence complementary to a part of the single-stranded DNA derived from the first primer (homologous to the template single-stranded DNA) was added to (D-4). ) Is hybridized with the single-stranded DNA derived from the first primer (addition of sense primer).
(D-6) A single-stranded DNA derived from the first primer and a single-stranded DNA derived from the second primer are extended by an enzyme having DNA-dependent DNA polymerase activity.
(D-7) The single-stranded DNA derived from the second primer is released from the single-stranded DNA derived from the first primer by the enzyme having the strand substitution activity and the single-stranded DNA derived from the extended first primer. ..
(D-8) The first primer is hybridized to the single-stranded DNA to which the primer released in (D-7) is added (addition of antisense primer).
(D-9) A double-stranded DNA having a promoter sequence added to the 5'end side is synthesized by an enzyme having DNA-dependent DNA polymerase activity.
An RNA polymerase corresponding to the promoter is allowed to act on the promoter-added double-stranded DNA synthesized in (D-10) and (D-9) to synthesize an RNA transcript (amplification of the RNA transcript).
(D-11) The second primer is hybridized to the RNA transcript (addition of sense primer).
(D-12) Synthesis of cDNA complementary to RNA transcript is performed by an enzyme having RNA-dependent DNA polymerase activity (synthesis of RNA-DNA double strand).
(D-13) The cDNA synthesized in (C-12) by an enzyme having ribonuclease H (RNase H) activity is converted into single-stranded DNA (degradation of RNA transcript).
RNA transcripts are continuously synthesized using the single-stranded DNA obtained in (D-14) and (D-13) as a template (amplification of RNA transcripts).

Next, the single-stranded nucleic acid to be amplified is a single-stranded DNA having a complementary region on the 3'end side and the other region (the region sandwiched between both complementary regions) does not have a complementary region. The flow of single-stranded nucleic acid amplification using the amplification reagent of the present invention when the primer to which the promoter sequence of the enzyme having RNA polymerase activity is added is the first primer will be described below (FIG. 6). In FIG. 6, as in FIG. 2, the amplification step by the TRC method (the steps after (6) below) is abbreviated as “TRC reaction”.

(1) Of the single-stranded DNA to be amplified (primer single-stranded DNA), it has a sequence complementary to a part of the region other than the complementary region, and RNA polymerase activity is on the 5'end side of the sequence. The first primer to which the promoter sequence of the enzyme having is added is hybridized with the single-stranded DNA (addition of antisense primer).
(2) The single-stranded DNA derived from the first primer and the template single-stranded DNA are extended by an enzyme having DNA-dependent DNA polymerase activity and an enzyme having strand substitution activity.
(3) The single-stranded DNA derived from the first primer is released from the template single-stranded DNA by an enzyme having strand substitution activity and an elongated template single-stranded DNA.
(4) The second primer having a sequence complementary to a part of the single-stranded DNA derived from the first primer (homologous to the template single-stranded DNA) was released in (3). Hybridize to single-stranded DNA derived from the first primer (addition of sense primer).
(5) A double-stranded DNA (TRC reaction complex) having a promoter sequence added to the 5'end side is synthesized by an enzyme having DNA-dependent DNA polymerase activity.
(6) An RNA polymerase corresponding to the promoter is allowed to act on the promoter-added double-stranded DNA synthesized in (5) to synthesize an RNA transcript (amplification of the RNA transcript).
(7) The second primer is hybridized to the RNA transcript (addition of sense primer).
(8) Synthesis of cDNA complementary to RNA transcript is performed by an enzyme having RNA-dependent DNA polymerase activity (synthesis of RNA-DNA double strand).
(9) The cDNA synthesized in (8) is converted into single-stranded DNA by an enzyme having ribonuclease H (RNase H) activity (degradation of RNA transcript).
(10) RNA transcripts are continuously synthesized using the single-stranded DNA obtained in (9) as a template (amplification of RNA transcripts).

Note that (A-9), (A-13), (B-9), (B-13), (C-10), (C-14), (D-10), (D-14), ( The fluorescence dye-labeled oligonucleotide probe designed to change the fluorescence characteristics by hybridizing (forming a complementary double strand) with a part of the RNA transcripts synthesized in 6) and (10) is amplified. When included in the system, the change in fluorescence characteristics can be measured over time with a fluorescence spectrophotometer to amplify and detect the target nucleic acid contained in the sample in one step and in a closed container.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

<Example 1> Synthesis of single-stranded DNA For the synthesis of single-stranded DNA, Long ssDNA Preparation Kit for 3.0 kb (manufactured by BioDynamics Laboratory) was used.

(1) pLSODN-3 (SEQ ID NO: 3) included in the kit was amplified by PCR using the primers of SEQ ID NO: 1 and SEQ ID NO: 2. Specifically, a reaction solution having the following composition was prepared in each of 16 PCR tubes with sterilized water so as to have a volume of 20 μL, and the temperature was 98 ° C. for 2 minutes (98 ° C. for 10 seconds, 65 ° C. for 5 seconds, 72 ° C. for 35 seconds). The reaction was carried out at 72 ° C. for 1 minute for 40 cycles.

Reaction solution composition:
1 x PrimeSTAR MAX Premix
0.2 μM F primer (SEQ ID NO: 1)
0.2 μM R primer (SEQ ID NO: 2)
1 ng Template (SEQ ID NO: 3)

The amplified PCR product was electrophoresed by 0.8% agarose gel electrophoresis to cut out a band of interest, and the QIAquick Gel Execution Kit (QIA).
Purified using (manufactured by GEN) to obtain a vector.

(2) Product L1 amplified by PCR using pAAV-CMV (SEQ ID NO: 4) using the primers of SEQ ID NO: 5 and SEQ ID NO: 6 and product L2 amplified using the primers of SEQ ID NO: 7 and SEQ ID NO: 6 Made. Specifically, a reaction solution having the following composition was prepared in each of eight PCR tubes so as to have a volume of 20 μL with sterile water, and was prepared at 98 ° C. for 2 minutes (98 ° C. for 10 seconds, 60.4 ° C. for 5 seconds, 72 ° C. for 12). The reaction was carried out at 72 ° C. for 1 minute for 40 cycles per second).

Reaction solution composition:
1 x PrimeSTAR MAX Premix
0.2 μM F primer (SEQ ID NO: 5 or 7)
0.2 μM R primer (SEQ ID NO: 6)
1 ng Template (SEQ ID NO: 4)

The amplified PCR product was electrophoresed by 0.8% agarose gel electrophoresis to cut out a band of interest, and the QIAquick Gel Execution Kit (QIA).
Purified using (manufactured by GEN) to obtain L1 or L2. Further, using L1 as a template and SEQ ID NO: 5 and SEQ ID NO: 8 and L2 as templates, the combinations of the primers of SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 8, SEQ ID NO: 7 and SEQ ID NO: 10 are used. PCR amplification was performed in the same manner as described above to obtain S2, S3, S4-2, and S5, respectively. In addition, S2, S3, and S5 amplified the PCR elongation at 72 ° C. for 15 seconds, and S4-2 amplified the PCR at 72 ° C. for 5 seconds. The amplified PCR product was electrophoresed on a 0.8% agarose gel electrophoresis to cut out a band of interest, purified using a QIAquick Gel Execution Kit (manufactured by QIAGEN), and inserted S2, S3, and S4-2, respectively. , S5.

(3) A plasmid was synthesized using the vector prepared in (1) and the inserts S2, S3, S4-2, and S5 prepared in (2) using the In Fusion reagent. About 50 ng of the vector and about 45 ng of the insert were used, In-Fusion HD Enzyme was added, and after a reaction at 50 ° C. for 15 minutes, a part was transformed into JM109. It was applied to an agar medium containing sodium carbenicillin and cultured at 37 ° C. at O / N. Colonies were cultured, plasmids were extracted and sequenced. Approximately 15 μg to 28 μg of plasmid containing the insert of interest was prepared.

(4) Nicks were inserted into the insert sites of each of the prepared plasmids using the nicking enzymes Nt BspQI and Nb BsrDI. After ethanol precipitation, Denaturing Gel-Loading buffer was added, and single-stranded DNA was released and electrophoresed by agarose gel electrophoresis. A band that seems to be single-stranded DNA was cut out and used as single-stranded DNA standard samples S2, S3, S4-2, and S5.

(5) The concentration of each single-stranded DNA was determined by absorbance measurement using NanoDrop, and the copy number was calculated from the base length.

<Example 2> Detection of single-stranded DNA by TRC reaction Various single-stranded DNAs (single-stranded DNA having a complementary region on the 3'end side and no other region having a complementary region) are TRC. Amplified by the reaction.
(1) S4-2 of single-stranded DNA (SEQ ID NO: 19; hereinafter, also simply referred to as standard DNA) is 10 using a TE buffer containing 0.01% sodium cholic acid and 0.01% sodium azide. ^It was diluted to ⁵ copies / 2 μL and used as a single-stranded DNA sample.

(2) After dispensing 10 μL of the reaction solution having the following composition into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), 2 μL of the DNA sample was added. A TaqMan probe (manufactured by IDT) was used as the standard DNA detection probe. Here, the first primer is an antisense primer with a promoter, and the second primer is a sense primer.

Composition of reaction solution: The concentration is the final concentration 60 mM Tris-HCl buffer (pH 8.65) after the addition of the initiator described later (in 20 μL).
0.3 mM dATP, dCTP, dGTP, dTTP, respectively
3.0 mM ATP, CTP, GTP, TTP respectively
3.4 mM ITP
67 mM trehalose 25 nM TaqMan probe (SEQ ID NO: 18)
1.0 μM first primer (SEQ ID NOs: 11-15)
1.0 μM second primer (SEQ ID NO: 16 or 17)
2U 96-7 DNA polymerase 4.3U AMV reverse transcriptase 95U T7 RNA polymerase

(3) The above reaction solution was kept warm at 46 ° C. for 3 minutes, and then 8 μL of an initiator having the following composition was added.
Initiator composition: The concentration is the final concentration after addition of the initiator (in 20 μL) 19.0 mM magnesium chloride 95.0 mM potassium chloride 3.8% (w / v) Glycerol
10.5% (v / v) DMSO

(4) Using a fluorescence spectrophotometer with a temperature control function that can directly measure the PCR tube, react at 46 ° C. and at the same time measure the fluorescence intensity (excitation wavelength 470 nm, fluorescence wavelength 520 nm) of the reaction solution over time for 30 minutes. did.
When the time when the initiator was added was 0 minutes, the case where the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a predetermined time by the fluorescence intensity ratio of the background) exceeded 1.8 was regarded as a positive determination. A positive judgment within 30 minutes was defined as (+), and a non-positive judgment (negative judgment) was defined as (-). The results are shown in Table 1.

It was found that S4-2 having a repetitive sequence at the 3'end can be amplified by SEQ ID NO: 11 to 15 when the second primer is SEQ ID NO: 16. Further, in the case of SEQ ID NO: 17, amplification was confirmed in combination with SEQ ID NO: 13. It was found that the selection of primers is important for detection in terms of amplification efficiency.

The present invention can be applied to reagents and the like.

Claims (5)

A single-stranded nucleic acid amplification reagent having at least a complementary region on the 3'terminal side containing at least the following (1) to (7) (provided that one of the following (6) and (7) is used. , The promoter sequence of the enzyme of (4) below is added to the 5'terminal side);
(1) An enzyme having RNA-dependent DNA polymerase activity;
(2) Enzyme having DNA-dependent DNA polymerase activity;
(3) Enzyme having ribonuclease H activity;
(4) Enzyme having RNA polymerase activity;
(5) Enzyme having chain substitution activity;
(6) A first primer having a sequence complementary to a part of a region other than the complementary region of the single-stranded nucleic acid; and (7) A region of the single-stranded nucleic acid other than the complementary region. A second primer having a sequence homologous to a portion of. The amplification reagent according to claim 1, wherein the (1), (2) and (3) are AMV reverse transcriptases. A method for amplifying a single-stranded nucleic acid having a complementary region at least on the 3'-terminal side, which comprises at least the following steps (I) to (VIII);
(I) Among the single-stranded nucleic acids, a first primer having a sequence complementary to a part of a region other than the complementary region, an enzyme having DNA-dependent DNA polymerase activity, and an enzyme having strand substitution activity. To synthesize DNA complementary to the single-stranded nucleic acid using
(II) A step of converting the DNA synthesized in (I) into a single-stranded DNA having a complementary region on the 3'end side using an enzyme having a strand substitution activity;
(III) Of the single-stranded nucleic acids, a second primer having a sequence homologous to a part of a region other than the complementary region, an enzyme having DNA-dependent DNA polymerase activity, and an enzyme having strand substitution activity. A step of synthesizing DNA homologous to the single-stranded nucleic acid using and.
(IV) A promoter of an enzyme having RNA polymerase activity possessed by either one of the first primer and the second primer by using the first primer and an enzyme having DNA-dependent DNA polymerase activity. A step of synthesizing double-stranded DNA from the DNA synthesized in (III) above with a sequence added;
(V) A step of synthesizing an RNA transcript from the double-stranded DNA using an enzyme having RNA polymerase activity corresponding to the promoter sequence;
(VI) Complementary to the RNA transcript using a primer having a sequence complementary to the RNA transcript among the first primer and the second primer and an enzyme having RNA-dependent DNA polymerase activity. Steps of synthesizing typical cDNA;
(VII) A step of converting the cDNA into a single-stranded DNA using an enzyme having ribonuclease activity; and (VIII) using the single-stranded DNA obtained in the above (VII) as a template, continuously producing RNA transcripts. The process of synthesizing. The step of synthesizing an RNA transcript using the single-stranded nucleic acid amplification method according to claim 3; and by forming a double strand complementary to a part of the RNA transcript, the fluorescence characteristics are improved as compared with those before formation. A method for detecting a single-stranded nucleic acid having a complementary region at least on the 3'end side, which comprises a step of detecting an RNA transcript using a changing oligonucleotide probe. A method of evaluating the quality of a sample containing a viral vector.
The single-stranded nucleic acid in the method for detecting a single-stranded nucleic acid according to claim 4 is a single-stranded nucleic acid of a viral vector, and the viral vector contained in the sample is obtained by using the method for detecting the single-stranded nucleic acid. A method comprising a step of qualitatively and / or quantitatively detecting and evaluating the quality of a sample containing the viral vector based on the detection result.

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