JP4406968B2 - Simple and enhanced nucleic acid amplification method - Google Patents

Description

【図面の簡単な説明】
【図１】実施例１の電気泳動結果を示した。標準マーカーとして１０¹²コピー／レーンの標準ＲＮＡ（２９８ヌクレオチド）、サイズマーカーとしてφＸ１７４／Ｈａｅ ＩＩＩを泳動した。特異増幅産物のバンドは標準ＲＮＡと同位置に現れる。特異産物と非特異産物のバンドの位置を矢印で示した。
【図２】実施例１の電気泳動結果で得られた特異産物のバンドの濃さをデンシトメーターで定量した結果を示した。
【図３】実施例２で行った測定結果を示した。グラフは、各反応時間における蛍光増加を示したものである。蛍光増加とは、各反応時間における蛍光強度からバックグラウンドの蛍光強度（反応時間０分の蛍光強度）を差し引いた値である。図中、バツ（×）は０コピー／テストの、黒丸（●）は１０⁴コピー／テストの、そして黒三角（▲）は１０⁴コピー／テストの結果をそれぞれ示す。
【図４】実施例３で行った測定結果を示した。グラフは、各反応時間に対する蛍光強度比を示したものである。蛍光強度比とは、バックグラウンドの蛍光強度（反応時間０分の蛍光強度）を１とした場合の各時間の蛍光強度の比である。
【図５】実施例４で行った測定結果を示した。グラフは、上から順に、０、２、４％ ＤＭＳＯ添加の場合の各反応時間における蛍光強度比を示したものである。蛍光強度比とは、バックグラウンドの蛍光強度（反応時間０分の蛍光強度）を１とした場合の各時間の蛍光強度の比である。黒菱形（◆）は陰性コントロール、白四角（□）は１０³コピー／テストのＲＮＡ標準、黒丸（●）は１０⁶コピー／テストのＲＮＡ標準を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for amplifying a target nucleic acid when analyzing a specific nucleic acid expected to be contained in a gene mixture such as DNA or RNA, and is useful for use in clinical diagnostic fields such as gene diagnosis, Furthermore, it is useful for qualitative or quantitative analysis of microorganisms present in environments such as food, indoors, soil, rivers, and the ocean.
[0002]
[Prior art]
Generally, in the diagnosis of viral infections, etc., the target nucleic acid (viral nucleic acid) in clinical samples is often very small, so the signal intensity is improved to achieve high sensitivity and good reproducibility measurement. For the purpose of increasing the sensitivity, a method of amplifying the target nucleic acid is used. In general, a polymerase chain reaction (PCR) method is known as a method for amplifying a specific DNA sequence. This method repeats a cycle consisting of thermal denaturation, primer annealing, and extension reaction in the presence of a pair of primers complementary to or homologous to both ends of the specific DNA sequence and a heat-resistant DNA polymerase. Is to amplify.
[0003]
On the other hand, as a method for amplifying a specific RNA sequence, a NASBA method, a 3SR method, or the like for amplifying a target RNA containing a specific RNA sequence by a concerted action of a reverse transcriptase and a DNA-dependent RNA polymerase is known. These methods synthesize a double-stranded DNA containing a promoter sequence with a primer containing a promoter sequence, reverse transcriptase, and ribonuclease H against a target RNA, and from a specific RNA sequence by DNA-dependent RNA polymerase. RNA is synthesized, and the RNA is subsequently subjected to a chain reaction that serves as a template for synthesizing double-stranded DNA containing a promoter sequence.
[0004]
In general, when RNA is amplified by PCR, RNA is used as a template, and cDNA is synthesized once by reverse transcriptase, and then PCR is performed, which is a substantially two-step process. There must be. Moreover, since PCR requires rapid temperature increase and decrease, there is a problem that a special incubator is required and it is not easy to apply to automation for mass processing.
[0005]
On the other hand, when a specific RNA sequence amplification method (NASBA method, 3SR method, or the like) is used, the reaction can be performed at a relatively constant temperature, and therefore, application to automation is easy. However, in order to detect a very small amount of a specific RNA sequence present in a sample with high sensitivity and good reproducibility, it is necessary to further improve the reaction efficiency.
[0006]
In these amplification methods, (1) the RNA portion (digested product of ribonuclease H) of the RNA-DNA hybrid produced as a result of cDNA synthesis is dissociated, and (2) non-specific products are reduced by binding of primers. For example, dimethylsulfoxide (DMSO) having a final concentration of about 15% is added.
[0007]
[Problems to be solved by the invention]
When DMSO is added excessively, the enzyme activity is reduced. From the viewpoint of enzyme activity, it is considered that the amplification efficiency can be improved when the DMSO concentration is as low as possible. However, in general, amplification methods of specific RNA sequences (NASBA method, 3SR method, etc.) are reacted at 41 ° C. Therefore, when the DMSO concentration is lowered, the above-mentioned (1) and (2) become poor, and amplification efficiency There was a problem that would decrease.
[0008]
Therefore, the object of the present invention is to achieve dissociation of the RNA portion (digested product of ribonuclease H) of the RNA-DNA hybrid produced as a result of cDNA synthesis and reduction of non-specific products by binding of primers, An object of the present invention is to provide an enhanced method for amplifying a specific nucleic acid, which can prevent a decrease in enzyme activity by not using a DMSO concentration or using only a lower concentration of DMSO than in the prior art.
[0009]
[Means for Solving the Problems]
As a result of intensive studies by the present inventors to achieve the above object, an RNA-DNA hybrid produced as a result of cDNA synthesis by performing an amplification reaction of a specific RNA sequence in a temperature range of 48 ° C. to 51 ° C. It is possible to reduce the non-specific product by the dissociation of the RNA portion (digested product of ribonuclease H) and the binding of the primers, thereby suppressing the DMSO concentration to a low concentration of 0 to 3% and reducing the enzyme activity. It has been found that the influence of DMSO can be reduced, and the present invention has been completed.
[0010]
That is, the present invention uses the specific RNA sequence as a template to generate a double-stranded DNA having a promoter sequence complementary to and homologous to the specific RNA sequence and capable of transcribing the sequence, and the DNA-dependent RNA polymerase In the RNA amplification method in which an RNA transcript of a specific sequence is generated and the RNA transcript is further used as a template for the double-stranded DNA, the reaction is carried out in a temperature range of 48 ° C. to 51 ° C. and 0 to 3% dimethyl. An enhanced method for amplifying a specific sequence, characterized in that the method is carried out in a sulfoxide (DMSO) concentration range.
[0011]
In the present invention, the specific RNA sequence or a target nucleic acid containing the specific RNA sequence is amplified by the above amplification method in the presence of an oligonucleotide probe labeled with an intercalating fluorescent dye, and the fluorescence intensity of the reaction solution is measured over time. A method for analyzing a specific nucleic acid sequence or a target nucleic acid containing a specific RNA sequence. Hereinafter, the present invention will be described in detail.
[0012]
In one embodiment of the present invention, at least the following reagents (A) to (I) are prepared in a sample expected to contain the target RNA, and these are sequentially added to the sample, or two or more of these are once added. Or adding them all at once ((A) to (I) do not need to be added in this order), and these reagents are added in the presence of DMSO at a final concentration of 0 to 3%. This is an enhanced method for amplifying a specific nucleic acid comprising a step of reacting at 48 to 51 ° C.
[0013]
(A) a first single-stranded oligonucleotide having a sequence complementary to the 3 ′ terminal sequence of the specific RNA sequence in the single-stranded RNA;
(B) RNA-dependent DNA polymerase,
(C) Deoxyribonucleoside triphosphate
(D) Ribonuclease H or an enzyme having an equivalent RNA degradation activity,
(E) In order from the 5 ′ end side (1) Promoter sequence of DNA-dependent RNA polymerase, (2) Enhancer sequence of the promoter, and (3) Same as 5 ′ end sequence of specific RNA sequence in the single-stranded RNA A second single-stranded oligonucleotide having the base sequence:
(F) DNA-dependent DNA polymerase,
(G) DNA-dependent RNA polymerase,
(H) Ribonucleoside triphosphate (adenosine triphosphate, uridine triphosphate, cytidine triphosphate, guanosine triphosphate)
(I) Inosine triphosphate
In another aspect of the present invention, in addition to the combination of the first and second single-stranded oligonucleotides of the reagent (A) and the reagent (E), the following reagents (J) and reagents (K) A combination of first and second single stranded oligonucleotides may be used.
[0014]
(J) In order from the 5 ′ end side: (1) the promoter sequence of DNA-dependent RNA polymerase, (2) the enhancer sequence of the promoter, and (3) the 3 ′ end sequence of the specific RNA sequence in the single-stranded RNA A first single-stranded oligonucleotide having a complementary sequence;
(K) a second single-stranded oligonucleotide having the same base sequence as the 5 ′ terminal sequence of the specific RNA sequence in the single-stranded RNA
The specific RNA sequence means that when the first and second single-stranded oligonucleotides of the above (A) and (E) are used, the 5 ′ end in the second single-stranded oligonucleotide described in (E) A base sequence portion present in the single-stranded RNA, beginning with the sequence (3) and ending with a sequence complementary to the first single-stranded oligonucleotide described in (A) at the 3 ′ end, When the first and second single-stranded oligonucleotides of (J) and (K) are used, the 5 ′ end begins with the same sequence as the second single-stranded oligonucleotide described in (K). 'A base sequence portion existing in the single-stranded RNA ending with a sequence complementary to the sequence of (3) in the first single-stranded oligonucleotide described in (J). The specific RNA sequence can be arbitrarily determined, but it is important to include a sequence portion specific enough to distinguish the target RNA from other nucleic acids.
[0015]
The reagent (A) is a first single-stranded oligonucleotide having a sequence complementary to the specific base sequence 3 ′ terminal sequence. The reagent complementarily binds to the target RNA, and when cDNA synthesis is performed using the RNA as a template by RNA-dependent DNA polymerase in the presence of the reagents (B) and (C) described later, This is intended to locate a sequence complementary to the 3 ′ end of the specific RNA sequence at the side end.
[0016]
Reagent (B) is an RNA-dependent DNA polymerase, and reagent (C) is deoxyribonucleoside triphosphate that serves as its substrate. In the presence of the reagents (A) to (C), a cDNA having a sequence complementary to the 3 ′ end of the specific RNA sequence in the target RNA at the 5 ′ end is synthesized. This cDNA is in a state of forming a DNA-RNA double strand with the template RNA.
[0017]
As the reagent (D), ribonuclease H having an activity of cleaving RNA in the DNA-RNA duplex can be used, or an enzyme having an equivalent RNA degradation activity can be used. A reverse transcriptase represented by avian myoblastoma virus reverse transcriptase (hereinafter referred to as AMV reverse transcriptase) has an activity of cleaving RNA in a DNA-RNA duplex, and this kind of enzyme is used. May be.
[0018]
Reagent (E) comprises, in order from the 5 ′ end, (1) a promoter sequence of DNA-dependent RNA polymerase, (2) an enhancer sequence of the promoter, and (3) a base sequence identical to the 5 ′ end sequence of the specific RNA sequence. Having a second single-stranded oligonucleotide. In the second oligonucleotide, the part (3) binds to the 3 ′ end of cDNA synthesized by the coexistence of the reagents (A) to (D). Therefore, due to the coexistence of the reagents (C) and (F), the cDNA is used as a template from the 3 ′ end of the second oligonucleotide, and at the same time, the second oligonucleotide is used as the template from the 3 ′ end of the cDNA. Each complementary strand is synthesized by sex DNA polymerase, and a complete double-stranded DNA having a transcribable promoter sequence is synthesized.
[0019]
Reagent (F) is a DNA-dependent DNA polymerase. Some reverse transcriptases represented by AMV reverse transcriptase have DNA-dependent DNA polymerase activity, and this type of enzyme may be used.
[0020]
Reagent (G) is a DNA-dependent RNA polymerase, and reagents (H) and (I) are ribonucleoside triphosphates and inosine triphosphates that serve as substrates. Double-stranded DNA synthesized by the coexistence of the reagents (C) and (E) and (F) has a promoter region of DNA-dependent RNA polymerase at the end. For this reason, in the presence of the reagents (G), (H) and (I), synthesis of single-stranded RNA comprising a specific RNA sequence is started immediately after the DNA synthesis. Specific examples of the DNA-dependent RNA polymerase in the reagent (G) include T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase and the like.
[0021]
Single-stranded RNA synthesized by the coexistence of the reagents (G) to (I) is RNA having a specific RNA sequence. Therefore, when these are synthesized and coexist with the reagents (A) to (F), the above-described series of reactions are repeated. As described above, in the present invention, double-stranded DNA having a promoter region at the end as described above is synthesized based on a very small amount of target RNA present in a sample, and this is a single-stranded RNA comprising a specific RNA sequence. Become a source of synthesis. The synthesized single-stranded RNA contributes to the synthesis of new double-stranded DNA, and as a result, the amount of single-stranded RNA comprising a specific RNA sequence increases dramatically with time.
[0022]
In another aspect of the present invention, in the amplification step of the specific nucleic acid performed at a reaction temperature of 48 ° C. to 51 ° C. and a DMSO concentration of 0 to 3%, a step of adding a reagent (L) and measuring the fluorescence intensity of the reaction solution is performed. A method for analyzing a specific nucleic acid.
[0023]
(L) a third single-stranded oligonucleotide having a sequence complementary to the specific RNA sequence and labeled so as to emit a measurable fluorescence signal when bound to a nucleic acid having the sequence
The reagent (L) is a third single-stranded oligonucleotide having a sequence complementary to a specific RNA sequence and labeled so as to emit a measurable fluorescence signal when bound to a nucleic acid having the sequence. . The third oligonucleotide may be a DNA to which, for example, an intercalating fluorescent dye is bound. The DNA portion is preferably 6 to 100 nucleotides, more preferably 10 to 30 nucleotides for specific analysis of a specific RNA sequence. Of course, it is important that the DNA portion is a sequence that is present in a specific RNA sequence and is complementary to a sequence portion that is sufficiently distinguishable from nucleic acids other than the target nucleic acid.
[0024]
When a DNA part forms a complementary bond with a single-stranded RNA consisting of a synthesized specific RNA sequence, the RNA-dependent DNA polymerase in the already added reagent (C) acts to extend from its 3 ′ end. It is preferable that a sequence that is not complementary to a specific RNA sequence is added to the 3 ′ end, or that the 3 ′ end is chemically modified.
[0025]
An intercalating fluorescent dye is one in which when a DNA portion forms a complementary bond with another nucleic acid, it is intercalated into a double-stranded portion to change the fluorescence characteristics. For this purpose, it can be exemplified that the intercalating fluorescent dye is bound to the DNA via an appropriate molecular length linker that does not interfere with the intercalation into the double-stranded portion. The linker is not particularly limited as long as it is a molecule that does not prevent the intercalating fluorescent dye from intercalating into the double-stranded part. In particular, a linker molecule selected from bifunctional hydrocarbons having functional groups at both ends is convenient and preferable for modification to oligonucleotides. In addition, for example, a commercially available reagent set (C6-Thiolmodifier, trade name, manufactured by Clontech) or the like can also be used.
[0026]
The intercalating fluorescent dye is not particularly limited as long as its fluorescence characteristics change by intercalating into double strands, for example, the fluorescence wavelength to be emitted fluctuates or the like. From the viewpoint of the above, those having the property of increasing the fluorescence intensity by intercalation are particularly preferable. More specifically, thiazole orange, oxazole yellow or derivatives thereof having particularly remarkable changes in fluorescence intensity can be exemplified as particularly preferred intercalating fluorescent dyes.
[0027]
The position where the intercalating fluorescent dye is bound to the DNA via the linker is not hindered from intercalating into the double strand of the intercalating fluorescent dye, such as the 5 ′ end, 3 ′ end or the center of the DNA. And there is no restriction | limiting in particular unless the complementary coupling | bonding of a DNA part and RNA is inhibited.
[0028]
The amount of single-stranded RNA synthesized by the reagents (G) to (I) using double-stranded DNA having a promoter sequence as a template increases with time.
[0029]
The reagent (L) is coexisted with at least the reagents (A) to (I) in a sample expected to contain the specific RNA sequence, and single-stranded RNA having an increasing specific RNA sequence is measured as a fluorescence signal. . Here, even when the synthesized single-stranded RNA and the third oligonucleotide of the reagent (L) form a complementary bond and emit a fluorescence signal, this RNA is not contained in the reagents (A) to (C). It became clear that it functions as a template in DNA synthesis in the coexistence. That is, in the present invention, in the coexistence state of each reagent, a series of phenomena of cDNA synthesis from RNA, double-stranded DNA synthesis, and RNA synthesis from double-stranded DNA occurs in the presence of the third oligonucleotide. The fluorescence intensity increases in proportion to the increase.
[0030]
Another aspect of the present invention is the analytical method carried out at a reaction temperature of 48 ° C. to 51 ° C. and a DMSO concentration of 0 to 3%, wherein the first and second reagents (A), (E) and (L) are first and second. And a combination of the first to third single-stranded oligonucleotides of the reagent (J) and the reagent (K) and the reagent (M) instead of the combination of the first and third single-stranded oligonucleotides. It is an analysis method.
[0031]
(M) a third sequence having a sequence identical to a specific RNA sequence in the single-stranded RNA and labeled so as to emit a measurable fluorescence signal when bound to a nucleic acid having a sequence complementary to the sequence. Single-stranded oligonucleotide
The reagent (J) comprises, in order from the 5 ′ end, (1) a promoter sequence of a DNA-dependent RNA polymerase, (2) an enhancer sequence of the promoter, and (3) 3 ′ side of a specific RNA sequence in the single-stranded RNA. A first single-stranded oligonucleotide having a sequence complementary to a terminal sequence. The part (3) of the reagent is complementarily bound to the 3 ′ end in the specific RNA sequence, and in the presence of the reagents (B), (C) and (D), as in the case of using the reagent (A). Thus, cDNA complementary to the target RNA is synthesized.
[0032]
The reagent (K) is a single-stranded oligonucleotide having a complementary sequence at the 5 ′ end in the specific RNA sequence of the cDNA. The reagent (K) is a double strand having an RNA polymerase promoter sequence after complementary binding to the 5 ′ end of the specific RNA sequence of the cDNA and then coexistence of the reagent (C) and the reagent (F). DNA is synthesized. Further, in the double-stranded DNA, the second single-stranded RNA complementary to the specific RNA sequence is synthesized by the coexistence of the reagents (G) to (H). Further, the presence of the reagent (I) greatly increases the amount of synthesis of the single-stranded RNA.
[0033]
Further, the 3 ′ end of the second RNA is complementarily bound to the reagent (K), and the second cDNA of the second RNA is synthesized in the presence of the reagents (B) to (D). The second cDNA is complementarily bound to the part (3) of the reagent (J), and double-stranded DNA containing a promoter sequence is synthesized by the reagent (C) and the reagent (F), and the reagents (G) to (I) ) To synthesize the second RNA. The second RNA, which is complementary to the synthesized specific RNA sequence, becomes a new double-stranded DNA synthesis source, and a series of reactions are repeated, resulting in a dramatic increase in the amount of the second RNA. Increase.
[0034]
The second RNA obtained here has a sequence complementary to a specific RNA sequence in the target RNA. The amount of the second RNA synthesized by coexisting the reagent (M), which is a third single-stranded oligonucleotide having the same sequence as the specific RNA sequence and labeled with an intercalating fluorescent dye The fluorescence intensity increases in proportion to
[0035]
The measurement of the fluorescence signal in the present invention is preferably performed with time from immediately after the addition of the reagents (A) to (I) and the reagent (L) or the reagent (M), or after a predetermined time has elapsed after the addition. The third DNA of reagent (L) or reagent (M) repeats binding and dissociation with the synthesized RNA in the process of synthesizing single-stranded RNA, but the fluorescence signal measured at the time of binding is Since the amount of RNA is reflected, it is possible to follow the increase in single-stranded RNA over time. The measurement itself may be continuous or intermittent at regular intervals. The fluorescent signal measuring device does not constitute a part of the present invention, but any device can be used as long as it can continuously or intermittently measure the fluorescent signal in one or more reaction tubes. May be.
[0036]
The measurement time of the fluorescence signal includes the time when the fluorescence intensity of the test section containing a certain amount of target RNA increases, and the time when a significant difference is seen from the test section not containing the target RNA. The time until the fluorescence intensity of the included test section becomes substantially constant is approximately 4 hours, and 1 hour under suitable conditions.
[0037]
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, this invention is not limited by these Examples.
[0038]
Example 1
The effect of DMSO addition (final concentration 14.3%) and non-addition in target nucleic acid amplification was examined.
[0039]
(1) From the 5 ′ side, the standard RNA is guanine, adenine, adenine, uracil, adenine, cytosine, adenine, adenine, followed by base numbers 11 to 300 of human hepatitis C virus RNA (Kato et al., Proc Natl.Sci., USA, 1990, 87, 9524-9528), and after quantification by ultraviolet absorption at 260 nm, RNA dilution (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 0. 5 × 10 using 5 U / μl RNase Inhibitor) ⁶ Dilute to 2 μl. For the negative control (Nega), only the RNA dilution was used.
[0040]
(2) 9.66 μl of the reaction solution having the following composition was dispensed into a PCR tube (Gene Amp Thin-Walled Reaction Tubes, manufactured by PerkinElmer), and 2 μl of the RNA sample was added thereto, and then 50 μl of mineral oil was overlaid thereon. . Then, it heated at 65 degreeC for 5 minute (s).
[0041]
Composition of reaction solution
62.1 mM Tris-acetate buffer (pH 8.1)
21.0 mM magnesium acetate
194.1 mM potassium acetate
12.4% Sorbitol
15.5 mM DTT
1.55 mM each of dATP, dCTP, dGTP, dTTP
0.31 μM first oligonucleotide (SEQ ID NO: 1)
A second oligonucleotide having an SP6 promoter sequence of 0.31 μM (SEQ ID NO: 2; in the oligonucleotide of SEQ ID NO: 2 the 5 'end to the 17th adenine is the SP6 promoter sequence, and the 18th guanine to the 25th (Up to the second adenine is an enhancer sequence)
22.2% (final concentration 14.3%) or 0% (no addition) DMSO
Volumetric distilled water
(3) The above reaction solution was kept at 44 ° C., 47 ° C., and 50 ° C. for 5 minutes, respectively, and then 2.59 μl of enzyme solution having the following composition was added.
[0042]
Composition of enzyme solution
8.1 U / μl AMV reverse transcriptase (Takara Shuzo)
33.0 U / μl SP6 RNA Polymerase (Takara Shuzo)
11.6 U / μl Ribonuclease inhibitor (Takara Shuzo)
0.016% bovine serum albumin
(4) After reacting each at 44, 47 and 50 ° C. for 5 minutes, 0.75 μl of 20 mM NTP aqueous solution (2.0 mM each of ATP, CTP, GTP, UTP) was added, and 44, 47, The reaction was carried out at 50 ° C. for 4 hours.
[0043]
(5) After removing 8.3 μl of the reaction solution and performing 2% agarose electrophoresis, the gel was stained with SYBR Green II (Takara Shuzo).
[0044]
The results of electrophoresis are shown in FIG. Moreover, the result of having quantified the blackening degree of the band of the specific product obtained by this result with the densitometer was shown in FIG. When the reaction temperature was 44 ° C., a band of a specific product was observed when DMSO was added and DMSO was not added. When the reaction temperature was 47 ° C. and 50 ° C., no specific product was generated when DMSO was added, and a remarkable specific product was observed when DMSO was not added. However, when DMSO was not added, significant non-specific products were also observed at 44 ° C. and 47 ° C. at the same time as specific products (FIG. 1). From this, it was shown that the addition of DMSO reduces the generation of non-specific products, but at the same time the amplification efficiency decreases. It was concluded that the amount of amplification product (band blackening) was highest at 50 ° C. and no DMSO added, and the amplification efficiency was improved compared to 44 ° C. and DMSO added (FIG. 2).
[0045]
Example 2
The effect of low concentration DMSO (final concentration 0 to 3%) on target nucleic acid amplification (reaction temperature 48, 50 ° C.) was examined.
[0046]
(1) Base numbers 113 to 267 of human hepatitis C virus RNA (Kato et al., Proc. Natl. Sci., USA, 1990, 87, 9524 to 9528) are used as standard RNA samples and quantified by absorption at 260 nm in the ultraviolet region. Then, using an RNA diluent (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 0.5 U / μl RNase Inhibitor), 10 ^Four Dilute to copy / 4 μl. For the negative control (Nega), only the RNA dilution was used.
[0047]
(2) 15.2 μl of the reaction solution having the following composition was dispensed into a PCR tube (Gene Amp Thin-Walled Reaction Tubes, manufactured by PerkinElmer), and 4 μl of the RNA sample was added thereto, and then 100 μl of mineral oil was overlaid thereon. .
[0048]
Composition of reaction solution
59.2 mM Tris-acetate buffer (pH 8.1)
13.2 mM magnesium acetate
130.2 mM potassium acetate
15.8% Sorbitol
19.7 mM DTT
1.0 mM each of dATP, dCTP, dGTP, dTTP
2.0 mM each ATP, CTP, GTP, UTP
2.5 mM ITP
0.4 μM first oligonucleotide (SEQ ID NO: 3)
A second oligonucleotide having an SP6 promoter sequence of 0.4 μM (SEQ ID NO: 4; in the oligonucleotide of SEQ ID NO: 4, from the 5 ′ end to the 17th adenine is the SP6 promoter sequence, and from the 18th guanine to the adenine Until is an enhancer sequence)
A third oligonucleotide labeled with 0.049 μM intercalating fluorescent dye (SEQ ID NO: 5; in the oligonucleotide of SEQ ID NO: 5, intercalatoricity between the 5th cytosine and the 6th guanine (The fluorescent dye is labeled, and its 3 ′ end is glycolic acid modified. Hereinafter, it is referred to as YO-271.)
3.9 U / μl Ribonuclease inhibitor (Takara Shuzo)
DMSO with various concentrations (0%, 2.0%, 4.0%, 5.9%), final concentrations of 0%, 1.0%, 2.0%, 3.0%
Volumetric distilled water
(3) The reaction solution was heated at 65 ° C. for 5 minutes.
[0049]
(4) After the above reaction solution was kept at 50 ° C. for 5 minutes, 10.8 μl of enzyme solution having the following composition, which was kept at 50 ° C. for 1 minute in advance, was added.
[0050]
Composition of enzyme solution
83.3 mM Tris-acetate buffer (pH 8.1)
18.5 mM magnesium acetate
182.4 mM potassium acetate
22.2% Sorbitol
3.9 U / μl AMV reverse transcriptase (Takara Shuzo)
15.7U / μl SP6 RNA Polymerase (Takara Shuzo)
0.28 mg / ml bovine serum albumin
(5) Using a fluorescence spectrophotometer with a temperature control function capable of directly measuring the PCR tube, the temperature is kept at 50 ° C., and the fluorescence intensity of the reaction solution in the PCR tube is 5 minutes at an excitation wavelength of 490 nm and a fluorescence wavelength of 510 nm. Measured at intervals.
[0051]
The result of subtracting the background fluorescence intensity from the fluorescence intensity at each point is shown in FIG. When the reaction temperature was 50 ° C., a remarkable increase in fluorescence was observed at a DMSO concentration of 0 to 2%. At DMSO concentrations of 0% and 1%, a rapid increase in fluorescence intensity (rise) was observed after 30 minutes of reaction time, but the rise at DMSO concentration of 2% was about 40 minutes. When the reaction temperature was 48 ° C., a remarkable increase in fluorescence was observed at a DMSO concentration of 1 to 3%. At any reaction temperature, increasing DMSO concentration tended to decrease amplification efficiency. From the above results, when the reaction temperature is set to 48 ° C. or 50 ° C., it is reasonable to set the DMSO concentration in the range of 0 to 3%, and the highest is especially at the reaction temperature of 50 ° C. and no DMSO addition condition. Amplification efficiency was shown to be obtained.
[0052]
Example 3
The reaction temperature in nucleic acid amplification without DMSO was examined.
[0053]
(1) Base numbers 113 to 267 of human hepatitis C virus RNA (Kato et al., Proc. Natl. Sci., USA, 1990, 87, 9524 to 9528) are used as standard RNA samples and quantified by absorption at 260 nm in the ultraviolet region. Then, using an RNA diluent (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 0.5 U / μl RNase Inhibitor), 10 ⁶ 10 ^Four Dilute to copy / 4 μl. For the control test group (Nega), only the RNA dilution was used.
[0054]
(2) 15.2 μl of the reaction solution having the following composition was dispensed into a PCR tube (Gene Amp Thin-Walled Reaction Tubes, manufactured by PerkinElmer), and 4 μl of the RNA sample was added thereto, and then 100 μl of mineral oil was overlaid thereon. .
[0055]
Composition of reaction solution
59.2 mM Tris-acetate buffer (pH 8.2)
13.2 mM magnesium acetate
118.7 mM potassium acetate
15.8% Sorbitol
19.7 mM DTT
1.0 mM each of dATP, dCTP, dGTP, dTTP
2.0 mM each ATP, CTP, GTP, UTP
2.0 mM ITP
0.4 μM first oligonucleotide (SEQ ID NO: 3)
Second oligonucleotide with 0.4 μM SP6 promoter sequence (SEQ ID NO: 4)
Third oligonucleotide labeled with 0.049 μM intercalating fluorescent dye (YO-271, SEQ ID NO: 5)
3.9 U / μl Ribonuclease inhibitor (Takara Shuzo)
Volumetric distilled water
(3) After incubating the above reaction solution at 50 ° C. for 5 minutes, 10.8 μl of enzyme solution having the following composition was added.
[0056]
Composition of enzyme solution
83.3 mM Tris-acetate buffer (pH 8.2)
18.5 mM magnesium acetate
166.7 mM potassium acetate
22.2% Sorbitol
3.9 U / μl AMV reverse transcriptase (Takara Shuzo)
15.8U / μl SP6 RNA Polymerase (Takara Shuzo)
0.27 μg / μl bovine serum albumin
Volumetric distilled water
(4) Using a fluorescence spectrophotometer with a temperature control function capable of directly measuring a PCR tube, the temperature is kept at 50 ° C., and the fluorescence intensity of the reaction solution in the PCR tube is 5 minutes at an excitation wavelength of 490 nm and a fluorescence wavelength of 510 nm. Measured at intervals.
[0057]
FIG. 4 shows the change over time in the fluorescence intensity ratio (fluorescence intensity value at each reaction time / background fluorescence intensity value).
[0058]
Although no significant increase in fluorescence was observed when DMSO was not added and the reaction temperature was 48 ° C to 51 ° C, the increase in fluorescence at 50 ° C was the highest.
[0059]
From the above, it was shown that when DMSO is not added, remarkable amplification efficiency is obtained at a reaction temperature of 48 ° C. to 51 ° C., but the highest amplification efficiency can be obtained by setting the reaction temperature to 50 ° C.
[0060]
Example 4
The effect of low concentration DMSO (final concentration 0 to 4%) on target nucleic acid amplification (reaction temperature 50 ° C.) was examined (when the first oligonucleotide having a promoter sequence was used).
[0061]
(1) Base numbers 113 to 267 of human hepatitis C virus RNA (Kato et al., Proc. Natl. Sci., USA, 1990, 87, 9524 to 9528) are used as standard RNA samples and quantified by absorption at 260 nm in the ultraviolet region. Then, using an RNA diluent (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 0.5 U / μl RNase Inhibitor), 10 ^Three And 10 ⁶ Dilute to copy / 4 μl. For the negative control (Nega), only the RNA dilution was used.
[0062]
(2) 15.2 μl of the reaction solution having the following composition was dispensed into a PCR tube (Gene Amp Thin-Walled Reaction Tubes, manufactured by PerkinElmer), and 4 μl of the RNA sample was added thereto, and then 100 μl of mineral oil was overlaid thereon. .
[0063]
Composition of reaction solution
59.2 mM Tris-acetate buffer (pH 8.1)
13.2 mM magnesium acetate
130.2 mM potassium acetate
15.8% Sorbitol
19.7 mM DTT
1.0 mM each of dATP, dCTP, dGTP, dTTP
2.0 mM each ATP, CTP, GTP, UTP
2.0 mM ITP
A first oligonucleotide having a 0.4 μM SP6 promoter sequence (SEQ ID NO: 6; in the oligonucleotide of SEQ ID NO: 6, from the 5 ′ end to the 17th adenine is the SP6 promoter sequence, and from the 18th guanine to the 25th (Up to the second adenine is an enhancer sequence)
0.4 μM second oligonucleotide (SEQ ID NO: 7)
Third oligonucleotide labeled with 0.049 μM intercalating fluorescent dye (SEQ ID NO: 8; in the oligonucleotide of SEQ ID NO: 8, intercalating between the 14th adenine and the 15th cytosine from the 5 ′ end) (The fluorescent dye is labeled, and its 3 ′ end is glycolic acid-modified, hereinafter referred to as YO-HC1b)
3.9 U / μl Ribonuclease inhibitor (Takara Shuzo)
DMSO with various concentrations (0%, 4.0%, 7.9%), final concentrations of 0%, 2.0%, 4.0%
Volumetric distilled water
(3) The reaction solution was heated at 65 ° C. for 5 minutes.
[0064]
(4) After the above reaction solution was kept at 50 ° C. for 5 minutes, 10.8 μl of enzyme solution having the following composition, which was kept at 50 ° C. for 1 minute in advance, was added.
[0065]
Composition of enzyme solution
83.3 mM Tris-acetate buffer (pH 8.1)
18.5 mM magnesium acetate
182.4 mM potassium acetate
22.2% Sorbitol
3.9 U / μl AMV reverse transcriptase (Takara Shuzo)
15.7U / μl SP6 RNA Polymerase (Takara Shuzo)
0.28 mg / ml bovine serum albumin
(5) Using a fluorescence spectrophotometer with a temperature control function capable of directly measuring the PCR tube, the temperature is kept at 50 ° C., and the fluorescence intensity of the reaction solution in the PCR tube is 5 minutes at an excitation wavelength of 490 nm and a fluorescence wavelength of 510 nm. Measured at intervals.
[0066]
FIG. 5 shows the change over time in the fluorescence intensity ratio (fluorescence intensity value at each reaction time / background fluorescence intensity value) in the presence of various concentrations of DMSO. 10 if the DMSO concentration is 0% ⁶ Only the copy / test showed a significant increase in fluorescence at about 20 minutes. 10 if the DMSO concentration is 2% ⁶ Copy / test takes about 20 minutes and 10 ^Three The copy / test showed a significant increase in fluorescence in 30-40 minutes. 10% for a DMSO concentration of 4% ⁶ The copy / test showed a significant increase in fluorescence at about 20 minutes. 10 ^Three The copy / test showed an increase in fluorescence in 30-40 minutes, but a small slope of increase in fluorescence was observed. From the above, even when the first oligonucleotide having a promoter sequence was used, nucleic acid amplification was observed under the conditions of a reaction temperature of 50 ° C. and a DMSO concentration of 0 to 4%, but the maximum was obtained at a DMSO concentration of 2%. Amplification efficiency was obtained.
[0067]
【The invention's effect】
As is clear from the above description, according to the present invention, the single-stranded RNA containing a specific RNA sequence in a sample is kept at a substantially constant temperature without repeating the operation of rapidly raising and lowering the temperature of the reaction solution. In a method of rapidly amplifying a specific RNA sequence originally present in a sample by only one manual operation performed at the start of the reaction, the reaction temperature is 48 ° C. to 51 ° C., and the DMSO concentration is 0 to 3%. By doing so, it becomes possible to dramatically improve the amplification efficiency.
[0068]
In the present invention, a double-stranded DNA having a promoter region of a DNA-dependent RNA polymerase at the terminal is synthesized based on the target RNA in the sample, and this becomes a synthesis source of a large amount of single-stranded RNA. Furthermore, the amount of synthesized single-stranded RNA increases dramatically, and in the step of measuring the increase in fluorescence due to the complementary binding of the probe labeled with the intercalating fluorescent dye to the generated single-stranded RNA, the fluorescence intensity By analyzing the process of increasing the amount of RNA, a method for measuring the initial RNA easily and quickly is provided.
[0069]
[0070]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the result of electrophoresis in Example 1. FIG. 10 as a standard marker ¹² Copy / lane standard RNA (298 nucleotides) and φX174 / Hae III were run as size markers. The band of the specific amplification product appears at the same position as the standard RNA. The positions of the specific product and non-specific product bands are indicated by arrows.
FIG. 2 shows the result of quantifying the density of the band of the specific product obtained from the electrophoresis result of Example 1 using a densitometer.
FIG. 3 shows the results of measurement performed in Example 2. The graph shows the increase in fluorescence at each reaction time. The increase in fluorescence is a value obtained by subtracting the background fluorescence intensity (fluorescence intensity at 0 minute reaction time) from the fluorescence intensity at each reaction time. In the figure, cross (×) is 0 copy / test, black circle (●) is 10 ^Four Copy / test and black triangle (▲) is 10 ^Four The copy / test results are shown respectively.
FIG. 4 shows the results of measurement performed in Example 3. The graph shows the fluorescence intensity ratio for each reaction time. The fluorescence intensity ratio is a ratio of fluorescence intensity at each time when the background fluorescence intensity (fluorescence intensity at 0 minute reaction time) is 1.
FIG. 5 shows the results of measurement performed in Example 4. The graph shows the fluorescence intensity ratio in each reaction time in the case of 0, 2, 4% DMSO addition in order from the top. The fluorescence intensity ratio is a ratio of fluorescence intensity at each time when the background fluorescence intensity (fluorescence intensity at 0 minute reaction time) is 1. Black diamonds (◆) are negative controls, white squares (□) are 10 ^Three Copy / test RNA standard, black circle (●) is 10 ⁶ Copy / test RNA standards are shown.

Claims (5)

Using a specific RNA sequence as a template, a double-stranded DNA comprising a promoter sequence that is complementary and homologous to the specific RNA sequence and capable of transcribing the sequence is generated, and RNA transcription of the specific sequence by DNA-dependent RNA polymerase In the RNA amplification method of generating a product and further using the RNA transcript as a template for the double-stranded DNA, at least (1) two primers each consisting of a complementary or homologous sequence to a specific RNA sequence One primer of which has a promoter sequence of DNA-dependent RNA polymerase on the 5 ′ end side, (2) RNA-dependent DNA polymerase, (3) DNA-dependent DNA polymerase, (4) DNA-dependent RNA Polymerase, (5) deoxyribonucleoside triphosphate, (6) ribonuclease In the presence of reoside triphosphate and a target RNA containing a specific RNA sequence, complementary to the specific RNA sequence at a constant temperature of 48 ° C. to 51 ° C. and within a concentration range of 1 to 3% dimethyl sulfoxide (DMSO) An enhanced method for amplifying a specific sequence , characterized by reacting for a time sufficient to obtain a homologous RNA amplification product . The amplification method according to claim 1 , wherein the RNA-dependent DNA polymerase and the DNA-dependent DNA polymerase are avian myeloblastosis virus reverse transcriptase. The amplification method according to claim 1 , wherein the DNA-dependent RNA polymerase is an RNA polymerase of phage SP6. A specific RNA sequence or a target nucleic acid containing a specific RNA sequence is amplified by the amplification method of claims 1 to 3 in the presence of an oligonucleotide probe labeled with an intercalating fluorescent dye, and the fluorescence intensity of the reaction solution is changed over time. A method for analyzing a specific RNA sequence or a target nucleic acid containing a specific RNA sequence, comprising measuring. In the oligonucleotide probe labeled with the intercalating fluorescent dye, the intercalating fluorescent dye portion is intercalated into a double-stranded portion produced by complementary binding between RNA produced by DNA-dependent RNA polymerase and the probe. 5. The analysis method according to claim 4 , wherein the analysis method has a property of being categorized.

Images