JP4639629B2 - Ribonucleic acid amplification reagent composition capable of reducing false positives and ribonucleic acid amplification method using the same - Google Patents
Ribonucleic acid amplification reagent composition capable of reducing false positives and ribonucleic acid amplification method using the same Download PDFInfo
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Description
本発明は、偽陽性を低減可能なリボ核酸増幅試薬組成、および該組成を用いたリボ核酸の増幅方法ならびに該方法を使用するための試薬またはキットに関する。 The present invention relates to a ribonucleic acid amplification reagent composition capable of reducing false positives, a ribonucleic acid amplification method using the composition, and a reagent or kit for using the method.
ポリメラーゼ・チェイン・リアクション（ＰＣＲ）法に代表される核酸増幅法は、特定の配列を有する核酸を短時間で特異的に検出する目的において優れた方法である。しかし、ＰＣＲ法による増幅の鋳型となる核酸はデオキシリボ核酸（ＤＮＡ）の形で提供される必要があるため、リボ核酸（ＲＮＡ）を対象とする場合は、直接、ＰＣＲ法によって増幅することはできない。ＲＮＡをＰＣＲ法により検出するためには、ＲＮＡが有する遺伝情報を逆転写活性を有する酵素などを用いてＤＮＡに変換する必要がある。このような逆転写とＰＣＲを組み合わせた方法においては、特にＲＮＡのみが検出対象である場合、特定の予防処理をしない限り、ＲＮＡ試料に混入したＤＮＡによって肯定的な結果が導かれることによる偽陽性が大きな問題となる。 The nucleic acid amplification method represented by the polymerase chain reaction (PCR) method is an excellent method for the purpose of specifically detecting a nucleic acid having a specific sequence in a short time. However, since the nucleic acid that serves as a template for amplification by the PCR method must be provided in the form of deoxyribonucleic acid (DNA), it cannot be directly amplified by the PCR method when targeting ribonucleic acid (RNA). . In order to detect RNA by the PCR method, it is necessary to convert genetic information contained in RNA into DNA using an enzyme having reverse transcription activity. In such a method combining reverse transcription and PCR, particularly when only RNA is a detection target, a positive result is obtained by a positive result being derived from DNA mixed in the RNA sample unless specific preventive treatment is performed. Is a big problem.
上記問題を解決するための１つの可能性は接合プライマーの使用である。接合プライマーは、ゲノムＤＮＡがＲＮＡに転写されるエキソン配列と、プロセシングによりＲＮＡに転写されないイントロン配列を含むことを利用し、その５'末端がエキソンの５'末端の配列に相補的で、その３'末端が隣接したエキソンの３'末端のヌクレオチドに相補的だが、ゲノム上の該２つのエキソンの間に位置するイントロンの３'末端のヌクレオチドに相補的でないヌクレオチドを含むように配列が選択されたオリゴヌクレオチドである。この場合、試料へのＤＮＡの混入による偽陽性は少なくとも部分的に回避できる。しかしながら、該方法では、イントロンを含まない遺伝子に対しては無効である。 One possibility to solve the above problem is the use of conjugation primers. The joining primer uses an exon sequence in which genomic DNA is transcribed into RNA and an intron sequence that is not transcribed into RNA by processing, and its 5 ′ end is complementary to a sequence at the 5 ′ end of the exon. The sequence was selected to include a nucleotide that is complementary to the nucleotide at the 3 'end of the exon adjacent to the end but not complementary to the nucleotide at the 3' end of the intron located between the two exons on the genome. It is an oligonucleotide. In this case, false positives due to DNA contamination in the sample can be at least partially avoided. However, this method is ineffective for genes that do not contain introns.
さらに、ゲノム上に存在する偽遺伝子は長さにおいて転写されたＲＮＡと同一であるため、ＰＣＲにおいて両者を区別することができないという同じ問題を提示する。このような場合、転写反応の前に試料をＤＮアーゼで処理することが有効である（例えば、非特許文献１および２、参照）。偽陽性を低減させるためのＤＮアーゼの使用に関しては、例えば反応試薬に添加する２価イオンとしてマグネシウムの代わりにマンガンイオンを存在させることでＤＮアーゼを効率的に作用させる、などの改良が行われている（例えば、特許文献１参照）。 Furthermore, since the pseudogene present on the genome is identical to the RNA transcribed in length, it presents the same problem that the two cannot be distinguished in PCR. In such a case, it is effective to treat the sample with DNase before the transcription reaction (see, for example, Non-Patent Documents 1 and 2). With regard to the use of DNase for reducing false positives, improvements have been made such as allowing DNase to act efficiently by the presence of manganese ions instead of magnesium as divalent ions added to the reaction reagent. (For example, refer to Patent Document 1).
しかし、従来公知のいずれの技術においても、試料をＤＮＡ分解活性を有する酵素と反応させた後、煩雑な抽出操作などにより該酵素を除去したＲＮＡ試料を逆転写反応に供している。すなわち、試料を入手してからリボ核酸の増幅を行うまでに煩雑な偽陽性予防処理を行う必要があり、これは研究や診断の現場において人的または時間的な効率を低下させる要因の一つとなり得る。 However, in any conventionally known technique, after a sample is reacted with an enzyme having DNA degrading activity, the RNA sample from which the enzyme has been removed by a complicated extraction operation or the like is subjected to a reverse transcription reaction. In other words, it is necessary to carry out complicated false positive prevention treatments from the acquisition of the sample to the amplification of ribonucleic acid, which is one of the factors that reduce human or temporal efficiency in the field of research and diagnosis. Can be.
本発明は、上記課題を解決するためになされたものであって、その目的とするところは、簡便に偽陽性を低減可能なリボ核酸増幅試薬組成、および該組成を用いたリボ核酸の増幅方法ならびに該方法を使用するための試薬またはキットを提供することである。 The present invention has been made to solve the above-described problems, and the object of the present invention is to provide a ribonucleic acid amplification reagent composition that can easily reduce false positives, and a ribonucleic acid amplification method using the composition. As well as providing reagents or kits for using the method.
本発明者らは、上記目的を達成するために種々鋭意検討したところ、試料に含まれるＤＮＡの分解（偽陽性予防処理）とリボ核酸の増幅を１工程で行うことが可能な試薬組成を見出した。
すなわち、本発明は以下のような構成からなる。
The present inventors have conducted various studies to achieve the above object, and as a result, have found a reagent composition that can decompose DNA contained in a sample (false positive prevention treatment) and amplify ribonucleic acid in one step. It was.
That is, the present invention has the following configuration.
（１）少なくとも一種類の酵素を含有することによりＤＮＡ分解活性および逆転写活性およびＤＮＡポリメラーゼ活性を有することを特徴とする、リボ核酸増幅用試薬組成。 (1) A reagent composition for amplifying ribonucleic acid, characterized by having at least one kind of enzyme and having DNA decomposing activity, reverse transcription activity and DNA polymerase activity.
（２）ＤＮＡ分解活性を有する酵素、および逆転写活性および／またはＤＮＡポリメラーゼ活性を有する少なくとも一種類の酵素を含有することを特徴とする、リボ核酸増幅用試薬組成。 (2) A ribonucleic acid amplification reagent composition comprising an enzyme having a DNA degrading activity and at least one enzyme having a reverse transcription activity and / or a DNA polymerase activity.
（３）少なくとも一種類の酵素を用いて１工程でリボ核酸を増幅することを特徴とするリボ核酸増幅用試薬組成であって、
（ａ）ＤＮＡ分解活性を有する酵素により試料に含まれるデオキシリボ核酸を分解し、
（ｂ）逆転写活性を有する酵素により試料に含まれるリボ核酸をデオキシリボ核酸に変換し、
（ｃ）ＤＮＡポリメラーゼ活性を有する酵素により標的ＲＮＡに相補的なデオキシリボ核酸を増幅するための成分を含有する試薬組成。
(3) A ribonucleic acid amplification reagent composition comprising amplifying ribonucleic acid in one step using at least one kind of enzyme,
(A) Degrading deoxyribonucleic acid contained in a sample with an enzyme having a DNA degrading activity;
(B) ribonucleic acid contained in the sample is converted into deoxyribonucleic acid by an enzyme having reverse transcription activity,
(C) A reagent composition containing a component for amplifying deoxyribonucleic acid complementary to a target RNA by an enzyme having DNA polymerase activity.
（４）ＤＮＡ分解活性を有する酵素がＤＮアーゼIであることを特徴とする、（１）から（３）のいずれかに記載のリボ核酸増幅用試薬組成。 (4) The reagent composition for ribonucleic acid amplification according to any one of (1) to (3), wherein the enzyme having DNA degrading activity is DNase I.
（５）マンガンイオンを含まないことを特徴とする、（１）から（４）のいずれかに記載のリボ核酸増幅用試薬組成。 (5) The reagent composition for ribonucleic acid amplification according to any one of (1) to (4), which does not contain manganese ions.
（６）反応容器中にＤＮＡ分解活性を有する酵素、逆転写活性および／またはＤＮＡポリメラーゼ活性を有する酵素を一緒に存在させ、１工程でリボ核酸を増幅することを特徴とするリボ核酸の増幅方法。 (6) A method for amplifying ribonucleic acid, wherein an enzyme having DNA degradation activity, an enzyme having reverse transcription activity and / or DNA polymerase activity are present together in a reaction vessel, and ribonucleic acid is amplified in one step. .
（７）少なくとも一種類の酵素を用いて１工程でリボ核酸を増幅することを特徴とするリボ核酸増幅方法であって、下記工程
（ａ）ＤＮＡ分解活性を有する酵素により試料に含まれるデオキシリボ核酸を分解し、
（ｂ）逆転写活性を有する酵素により試料に含まれるリボ核酸をデオキシリボ核酸に変換し、
（ｃ）ＤＮＡポリメラーゼ活性を有する酵素により標的ＲＮＡに相補的なデオキシリボ核酸を増幅することから成る方法。
(7) A ribonucleic acid amplification method comprising amplifying ribonucleic acid in one step using at least one kind of enzyme, wherein the following step (a) deoxyribonucleic acid contained in a sample by an enzyme having DNA degrading activity Disassemble
(B) ribonucleic acid contained in the sample is converted into deoxyribonucleic acid by an enzyme having reverse transcription activity,
(C) A method comprising amplifying deoxyribonucleic acid complementary to a target RNA by an enzyme having DNA polymerase activity.
（８）ＤＮＡ分解活性を有する酵素がＤＮアーゼIであることを特徴とする、（６）または（７）に記載のリボ核酸増幅方法。 (8) The ribonucleic acid amplification method according to (6) or (7), wherein the enzyme having DNA-degrading activity is DNase I.
（９）マンガンイオンを含まないことを特徴とする、（６）から（８）のいずれかに記載のリボ核酸増幅用試薬組成。 (9) The reagent composition for ribonucleic acid amplification according to any one of (6) to (8), which does not contain manganese ions.
（１０）（６）から（９）のいずれかに記載の方法を用いてリボ核酸を増幅するための試薬またはキット。 (10) A reagent or kit for amplifying ribonucleic acid using the method according to any one of (6) to (9).
本発明により、試料に含まれるデオキシリボ核酸の分解（偽陽性予防処理）とリボ核酸の増幅を１工程で行うことができ、特に研究や診断の現場において人的または時間的な効率を大きく向上させることが可能となる。また、本発明の波及効果として、該現場において大きな問題となっている、増幅反応の途中で容器の蓋を開閉することによるコンタミネーションの予防策を提供することができる。 According to the present invention, deoxyribonucleic acid contained in a sample can be decomposed (false positive prevention treatment) and ribonucleic acid can be amplified in one step, which greatly improves human or temporal efficiency particularly in the field of research and diagnosis. It becomes possible. Further, as a ripple effect of the present invention, it is possible to provide a preventive measure against contamination by opening and closing the lid of the container during the amplification reaction, which is a major problem in the field.
本発明においてリボ核酸の増幅とは、試料中のリボ核酸から増幅したい核酸（標的核酸）をその固有の配列の相補性を利用して増幅することを指し、具体的には、該リボ核酸を自身に相補的なデオキシリボ核酸（ｃＤＮＡ）に変換した後、該ｃＤＮＡを増幅する一連の流れを指す。本発明におけるリボ核酸増幅試薬組成およびそれを用いたリボ核酸の増幅方法が適用可能な該ｃＤＮＡ増幅の方法としては、ＰＣＲ法、ＬＡＭＰ法、ＳＤＡ法など特に限定されるものではないが、一般的に熱安定性の高い酵素を用いるＰＣＲ法の使用がより好ましい。 In the present invention, amplification of ribonucleic acid refers to amplification of a nucleic acid to be amplified from a ribonucleic acid in a sample (target nucleic acid) using complementarity of its inherent sequence. Specifically, the ribonucleic acid is It refers to a series of streams in which the cDNA is amplified after being converted to deoxyribonucleic acid (cDNA) complementary to itself. The cDNA amplification method to which the ribonucleic acid amplification reagent composition and the ribonucleic acid amplification method using the same can be applied is not particularly limited, such as PCR method, LAMP method, SDA method, etc. It is more preferable to use a PCR method using an enzyme having high thermostability.
RNAを含む試料に混在するDNAによる擬陽性を防止するためには、まずRNAを含む試料に混在するDNAをDNアーゼで分解するが、次のRNAからｃDNAに変換した際にこのDNアーゼが残存している場合にはｃDNAが分解すると考えられていた。しかしながら発明者らの検討によると、逆転写酵素によりｃDNAに変換した際のRNA-ｃDNAハイブリッド状態では、DNアーゼが存在していても分解されることなくｃDNAが維持されることを見出し、さらに、引き続くPCR反応の高温下によってDNアーゼ、逆転写酵素の活性を失活させることができることにをヒントに、各酵素の組み合わせ、各酵素量の最適化、各酵素のいずれもが有効に働く試薬液組成の最適化を検討することにより、本発明を完成させた。 In order to prevent false positives due to DNA mixed in a sample containing RNA, DNA mixed in the sample containing RNA is first decomposed with DNase, but this DNase remains when the next RNA is converted to cDNA. The cDNA was thought to be degraded. However, according to the study by the inventors, it was found that in the RNA-cDNA hybrid state when converted to cDNA by reverse transcriptase, cDNA is maintained without being degraded even if DNase is present. With the hint that DNase and reverse transcriptase activities can be inactivated by the subsequent high temperature of the PCR reaction, the combination of each enzyme, the optimization of each enzyme amount, and the reagent solution in which each enzyme works effectively The present invention was completed by examining the optimization of the composition.
本発明の偽陽性を低減可能なリボ核酸増幅試薬組成の好ましい一例としては、まず、（ａ）ＤＮＡ分解活性を有する酵素、（ｂ）逆転写活性を有する酵素、（ｃ）ＤＮＡポリメラーゼ活性を有する酵素、を含むことが好ましい。
さらに、（ｄ）ＲＮアーゼ阻害剤、（ｅ）核酸増幅用プライマー、（ｆ）ｄＮＴＰ、（ｇ）反応バッファー、（ｈ）リボ核酸含有試料などを含むことができる。
As a preferred example of the ribonucleic acid amplification reagent composition capable of reducing false positives of the present invention, first, (a) an enzyme having a DNA degradation activity, (b) an enzyme having a reverse transcription activity, and (c) having a DNA polymerase activity It preferably contains an enzyme.
Further, (d) an RNase inhibitor, (e) a nucleic acid amplification primer, (f) dNTP, (g) a reaction buffer, (h) a ribonucleic acid-containing sample, and the like can be included.
（ａ）ＤＮＡ分解活性を有する酵素としては、各種のＤＮＡ依存的エキソヌクレアーゼまたはＤＮＡ依存的エンドヌクレアーゼを使用し得るが、ＤＮＡ−ＲＮＡハイブリッド２本鎖を分解する活性を有しない酵素の使用が望ましい。具体的にはＤＮアーゼの使用が好ましく、ＤＮアーゼIの使用がより好ましい。該酵素の使用量としては、おおよそ０〜２５℃程度の温度範囲にて６０分以内に偽陽性の原因となる試料中のデオキシリボ核酸を分解し、かつ後工程の酵素反応を阻害しない量であることが必要である。また、RNA-ｃDNAハイブリッドを実質上分解しない量であること、加熱のみで失活させうる量であることが大切である。具体的には、ＤＮアーゼIの場合は５〜５０００単位／ｍｌ程度の使用が好ましく、１００〜１０００単位／ｍｌ程度の使用がより好ましい。 (A) As an enzyme having a DNA degrading activity, various DNA-dependent exonucleases or DNA-dependent endonucleases can be used, but it is desirable to use an enzyme that does not have an activity of degrading a DNA-RNA hybrid duplex. . Specifically, the use of DNase is preferred, and the use of DNase I is more preferred. The amount of the enzyme used is an amount that decomposes deoxyribonucleic acid in the sample that causes false positives within 60 minutes in a temperature range of about 0 to 25 ° C. and does not inhibit the subsequent enzymatic reaction. It is necessary. In addition, it is important that the RNA-cDNA hybrid is in an amount that does not substantially decompose and that it can be deactivated only by heating. Specifically, in the case of DNase I, the use of about 5 to 5000 units / ml is preferred, and the use of about 100 to 1000 units / ml is more preferred.
（ｂ）逆転写活性を有する酵素としては、従来公知のレトロウイルス、特にモロニーマウス白血病ウイルス（Ｍ−ＭＬＶ）、ヒト後天性免疫不全ウイルス（ＨＩＶ）、トリ骨髄芽症ウイルス（ＡＭＶ）などに由来する各種の逆転写酵素を特に限定されることなく使用できる。また、レトロウイルス由来の逆転写酵素の多くでＤＮＡ−ＲＮＡハイブリッド２本鎖のＲＮＡを分解する活性、すなわちＲＮａｓｅＨ活性を有することが知られており、この活性の存在はＲＮＡを鋳型としてｃＤＮＡを合成する際に鋳型−プライマー複合体の鋳型を分解し、その分解位置がプライマーの３'端に近い場合は鋳型−プライマー複合体が解離されるため伸長性が低下するという結果を招く。このような問題を排除するため、本発明の逆転写活性を有する酵素としては、実質的にＲＮａｓｅＨ活性を有していない逆転写酵素を使用することが好ましい。具体的には、Ｍ−ＭＬＶ由来のＲＮａｓｅＨ活性除去変異体の場合は５〜５０００単位／ｍｌ程度の使用が好ましく、１００〜１０００単位／ｍｌ程度の使用がより好ましい。 (B) The enzyme having reverse transcription activity is derived from a conventionally known retrovirus, particularly Moloney murine leukemia virus (M-MLV), human acquired immunodeficiency virus (HIV), avian myeloblastosis virus (AMV), etc. Various reverse transcriptases can be used without any particular limitation. In addition, it is known that many retrovirus-derived reverse transcriptases have the activity of degrading DNA-RNA hybrid double-stranded RNA, that is, RNase H activity. The presence of this activity synthesizes cDNA using RNA as a template. In this case, the template of the template-primer complex is decomposed, and when the decomposition position is close to the 3 ′ end of the primer, the template-primer complex is dissociated, resulting in a decrease in extensibility. In order to eliminate such a problem, it is preferable to use a reverse transcriptase having substantially no RNase H activity as the enzyme having reverse transcription activity of the present invention. Specifically, in the case of an RNase H activity-removed mutant derived from M-MLV, the use of about 5 to 5000 units / ml is preferred, and the use of about 100 to 1000 units / ml is more preferred.
（ｃ）ＤＮＡポリメラーゼ活性を有する酵素としては、Ｔ４またはＴ７ファージ、大腸菌、サーモコッカス（Ｔｈｅｒｍｏｃｏｃｃｕｓ）属、サーマス（Ｔｈｅｒｍｕｓ）属、パイロコッカス（Ｐｙｒｏｃｏｃｃｕｓ）属、バシラス（Ｂａｃｉｌｌｕｓ）属など種々の起源のＤＮＡポリメラーゼおよびその改良変異体を特に限定されることなく使用できるが、核酸の増幅方法としてＰＣＲ法を使用する場合はＫＯＤ ＤＮＡポリメラーゼ、Ｔａｑ ＤＮＡポリメラーゼ、Ｔｔｈ ＤＮＡポリメラーゼ、Ｐｆｕ ＤＮＡポリメラーゼなど熱安定性の酵素を使用することが好ましい。中でもＫＯＤ ＤＮＡポリメラーゼまたは該酵素を改良したＤＮＡポリメラーゼは増幅量および反応速度の観点から最も好ましい。具体的には、ＫＯＤ Ｄａｓｈ ＤＮＡポリメラーゼの場合は１〜２００単位／ｍｌ程度の使用が好ましく、１０〜１００単位／ｍｌ程度の使用がより好ましい。
なお、（ｂ）逆転写活性を有する酵素および（ｃ）ＤＮＡポリメラーゼ活性を有する酵素については逆転写活性を有するＤＮＡポリメラーゼによる代替も可能であり、このような性質の酵素としてはＴｔｈ ＤＮＡポリメラーゼが最も代表的である。
(C) As an enzyme having DNA polymerase activity, DNA of various origins such as T4 or T7 phage, Escherichia coli, Thermococcus genus, Thermos genus, Pyrococcus genus, Bacillus genus, etc. Polymerase and improved variants thereof can be used without any particular limitation. When PCR is used as a nucleic acid amplification method, a thermostable enzyme such as KOD DNA polymerase, Taq DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, etc. Is preferably used. Of these, KOD DNA polymerase or a DNA polymerase obtained by improving the enzyme is most preferable from the viewpoints of amplification amount and reaction rate. Specifically, in the case of KOD Dash DNA polymerase, the use of about 1 to 200 units / ml is preferred, and the use of about 10 to 100 units / ml is more preferred.
Note that (b) an enzyme having reverse transcription activity and (c) an enzyme having DNA polymerase activity can be replaced by a DNA polymerase having reverse transcription activity, and Tth DNA polymerase is the most suitable enzyme having such properties. Representative.
本発明において、１工程で反応を行うためには、上述の各酵素の組み合わせの相性も考慮することが重要である。具体的な個々の酵素を選択するときには、各酵素が有効に作用するバッファー組成（ｐH、塩濃度等）が共通するもの、近いものを組み合わせることが好ましい。 In the present invention, in order to carry out the reaction in one step, it is important to consider the compatibility of the combinations of the aforementioned enzymes. When selecting specific individual enzymes, it is preferable to combine those having similar or similar buffer compositions (pH, salt concentration, etc.) in which each enzyme acts effectively.
（ｄ）ＲＮアーゼ阻害剤としては、ヒト胎盤やブタ肝臓などに由来する各種のＲＮアーゼ阻害剤を特に限定されることなく使用でき、５〜５０００単位／ｍｌ程度の使用が好ましく、１００〜１０００単位／ｍｌ程度の使用がより好ましい。 (D) As the RNase inhibitor, various RNase inhibitors derived from human placenta, pig liver and the like can be used without particular limitation, and the use of about 5 to 5000 units / ml is preferable, and 100 to 1000 Use of about unit / ml is more preferable.
（ｅ）核酸増幅用プライマーは１〜１０００ｎＭ程度を含む。中でも特異性を高めるために、１００〜５００ｎＭ程度がより好ましく、２００ｎＭ程度がさらに好ましい。 (E) The nucleic acid amplification primer contains about 1-1000 nM. In particular, in order to increase specificity, about 100 to 500 nM is more preferable, and about 200 nM is more preferable.
（ｆ）ｄＮＴＰは５０〜１０００μＭ程度を含む。１００〜７００μＭ程度がより好ましく、２００〜４００μＭ程度がさらに好ましい。
（ｇ）反応バッファーについては、本発明のリボ核酸増幅試薬に含まれる酵素の能力を最大限に発揮する目的で、緩衝液や金属塩の濃度、溶液のｐＨなどが適宜選択され得る。
これらは、組み合わせる酵素により、最適な範囲は異なるが、例えば実施例で示したＤＮａｓｅI（ロシュ製）、Ｍ−ＭＬＶ由来逆転写酵素ＲＮａｓｅＨ活性除去変異体（東洋紡績製）、熱安定性ＤＮＡポリメラーゼＫＯＤ Ｄａｓｈ（東洋紡績製）の組み合わせの場合では、ｐＨは室温下で測定した場合に６．５〜９．５、さらには室温下で測定した場合に８．０〜９．０が好ましく、Ｍｇ濃度は１．０ｍＭ〜５．０ｍＭ、さらには１．５〜４．０ｍＭが好ましい。
(F) dNTP contains about 50-1000 μM. About 100 to 700 μM is more preferable, and about 200 to 400 μM is more preferable.
(G) Regarding the reaction buffer, the concentration of the buffer solution or metal salt, the pH of the solution, etc. can be appropriately selected for the purpose of maximizing the ability of the enzyme contained in the ribonucleic acid amplification reagent of the present invention.
The optimum range of these varies depending on the enzyme to be combined. For example, DNaseI (Roche), M-MLV-derived reverse transcriptase RNaseH activity-removed mutant (Toyobo), thermostable DNA polymerase KOD shown in Examples In the case of a combination of Dash (manufactured by Toyobo), the pH is preferably 6.5 to 9.5 when measured at room temperature, and more preferably 8.0 to 9.0 when measured at room temperature. Is preferably 1.0 mM to 5.0 mM, more preferably 1.5 to 4.0 mM.
特に、１工程で反応を行うためには、試薬に含まれる酵素のいずれもがその能力を発揮できる試薬組成に調整する必要がある。特にMgは各酵素の依存性が高いため、各酵素の適正濃度範囲にすることが好ましい。なお、やむを得ない場合には反応中に塩などを追加しても良いが、操作性等の面で、反応開始時にはすべての添加物が反応溶液中に含まれる様にすることが好ましい。
また、酵素の安定性を高める目的で、該試薬にウシ血清アルブミンなどの添加剤を適宜混合しても良い。
In particular, in order to carry out the reaction in one step, it is necessary to adjust the reagent composition so that any enzyme contained in the reagent can exhibit its ability. In particular, since Mg is highly dependent on each enzyme, it is preferable to set the concentration within the appropriate concentration range for each enzyme. If unavoidable, salt or the like may be added during the reaction, but from the viewpoint of operability and the like, it is preferable that all additives are included in the reaction solution at the start of the reaction.
Further, for the purpose of enhancing the stability of the enzyme, an additive such as bovine serum albumin may be appropriately mixed with the reagent.
さらに、試薬組成物中のマンガンイオンの含有量は１ｍＭ以下であることが好ましい。マンガンイオンは通常、例えばＴｔｈ ＤＮＡポリメラーゼに代表される熱安定性ＤＮＡポリメラーゼの逆転写活性を増強するために添加することがあるが、マンガンイオンの存在は非特異的な増幅やスメア状の増幅を引き起こしやすくなる、または核酸を増幅する効率が低下するなどの理由で好ましくない。
マンガンイオンの含有量はさらに好ましくは０．５ｍＭ以下であり、特に好ましくは０．１ｍＭ以下であり、 最も好ましくは実質的に含有されないことである。実質的に含有されないとは、非特異的な増幅やスメア状の増幅を引き起こしやすくなる、または核酸を増幅する効率が低下するなどの不具合を生じない量以下である。
Furthermore, the manganese ion content in the reagent composition is preferably 1 mM or less. Manganese ions are usually added to enhance the reverse transcription activity of thermostable DNA polymerases such as Tth DNA polymerase. However, the presence of manganese ions causes nonspecific amplification and smear-like amplification. This is not preferable because it tends to be caused or the efficiency of amplifying nucleic acid decreases.
The manganese ion content is more preferably 0.5 mM or less, particularly preferably 0.1 mM or less, and most preferably not substantially contained. The term “substantially not contained” refers to an amount that does not cause inconveniences such as non-specific amplification and smear-like amplification, or a decrease in nucleic acid amplification efficiency.
（ｈ）リボ核酸は本発明において、例えば、ｍＲＮＡ、ｔＲＮＡ、ｒＲＮＡなどの、いずれのリボ核酸であってもよい。また本発明は、イントロンを含まないｍＲＮＡ試料、および偽遺伝子が存在するまたは偽遺伝子が存在するかどうかわからないｍＲＮＡ試料において、特に効果的に作用する。本発明によれば、偽陽性の原因となるデオキシリボ核酸が高度に混入した場合でさえ、リボ核酸を選択的に増幅し得る（実施例１を参照）。したがって、本発明のリボ核酸増幅方法において、完全には精製されていないＲＮＡを含有する試料を用いることが可能である。特にデオキシリボ核酸の含有量は、標的となるリボ核酸に対応するかどうかに関わらず、リボ核酸を増幅するための従来技術にしたがって用いられた試料中の含有量を越えることができる。試料は、例えば、バクテリア、動物または植物組織、個体細胞由来の溶解物などのあらゆる材料から調製することができる。また、リボ核酸は試料中に溶解させてもよいし、固相に固定させてもよい。 (H) In the present invention, the ribonucleic acid may be any ribonucleic acid such as mRNA, tRNA, and rRNA. The present invention also works particularly effectively on mRNA samples that do not contain introns and mRNA samples that contain pseudogenes or do not know whether pseudogenes are present. According to the present invention, ribonucleic acid can be selectively amplified even when deoxyribonucleic acid causing false positives is highly contaminated (see Example 1). Therefore, in the ribonucleic acid amplification method of the present invention, it is possible to use a sample containing RNA that has not been completely purified. In particular, the content of deoxyribonucleic acid can exceed the content in the sample used according to the prior art for amplifying ribonucleic acid, regardless of whether it corresponds to the target ribonucleic acid. The sample can be prepared from any material such as, for example, bacteria, animal or plant tissue, lysates from individual cells. Moreover, ribonucleic acid may be dissolved in a sample, or may be fixed to a solid phase.
増幅反応は、ＤＮＡ分解反応、逆転写反応、ＰＣＲ反応を含む。これらの反応は１ステップで行われることが好ましい。ここで言う１工程とは、これらの反応を途中に抽出・分離工程を入れることなく行うことであり、さらには途中で添加物等を加えることなく行われる行われることが好ましい。特には、測定サンプルを含めた反応溶液を調整した後は、温度コントロールおよび必要に応じて攪拌のみで各反応が順次進められることが好ましい。 The amplification reaction includes a DNA degradation reaction, a reverse transcription reaction, and a PCR reaction. These reactions are preferably carried out in one step. The one step referred to here is to perform these reactions without an extraction / separation step in the middle, and it is preferably performed without adding an additive or the like in the middle. In particular, after preparing the reaction solution including the measurement sample, it is preferable that each reaction is sequentially advanced only by temperature control and, if necessary, stirring.
以下に、ＤＮＡ分解活性を有する酵素としてＤＮアーゼIを、逆転写活性を有する酵素としてＭ−ＭＬＶ由来のＲＮａｓｅＨ活性除去変異体（東洋紡績製）を、ＤＮＡポリメラーゼ活性を有する酵素としてＫＯＤ Ｄａｓｈ（東洋紡績製）をそれぞれ用いることを例に挙げて、本発明について具体的に説明する。なお、該発明の最大の新規性は、増幅反応系からＤＮＡ分解活性を有する酵素を除去する工程および途中で酵素を含有する溶液などを添加する工程を省略し、１工程で、偽陰性の原因となり得るＤＮＡの分解から標的ＲＮＡに相補的なＤＮＡ増幅の反応までを行うことにある。したがって、以下に開示する例において構成成分の一部、すなわち酵素の変更または該変更に合わせた試薬組成の改良などは、前述の指針に従えば当業者にとって容易である。したがって、以下の事例は、本発明の内容を何ら限定するものではない。 In the following, DNase I is used as an enzyme having DNA-degrading activity, RNaseH activity-removing mutant derived from M-MLV (manufactured by Toyobo) as enzyme having reverse transcription activity, and KOD Dash (Toyobo) as enzyme having DNA polymerase activity. The present invention will be described in detail by taking as an example the use of each of (made by spinning). The greatest novelty of the invention is that the step of removing the enzyme having DNA degrading activity from the amplification reaction system and the step of adding a solution containing the enzyme in the middle are omitted, and the cause of false negatives is obtained in one step. The purpose is to perform from the decomposition of the potential DNA to the DNA amplification reaction complementary to the target RNA. Accordingly, in the examples disclosed below, it is easy for those skilled in the art to change some of the constituent components, that is, change the enzyme or improve the reagent composition in accordance with the change. Therefore, the following examples do not limit the contents of the present invention.
実施例１
本発明のリボ核酸増幅試薬組成における偽陽性低減効果の確認
（１）試料の調製
ＨｅＬａ細胞よりＲＮＡおよびＤＮＡをそれぞれ抽出した。該核酸を１％アガロースゲルにて電気泳動し、エチジウムブロマイド染色した後、紫外線照射下での蛍光を検出した。このとき核酸抽出に用いた細胞数および電気泳動に用いた核酸の量を表１に示し、該電気泳動によって得られた写真を図１に示す。なお、図１のレーン番号は表１のサンプル番号に対応する。電気泳動の条件は、定電圧１００Ｖ、３０分間にて行った。反応液の他に分子量マーカーも同時に泳動し、検出されたＤＮＡ断片の鎖長を比較する際の参考とした。操作方法ならびに他の条件は、マニアティス（Ｍａｎｉａｔｉｓ）らのモレキュラー・クローニング（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ）（１９８２年）に記載の技法に従った。
Example 1
Confirmation of false positive reduction effect in ribonucleic acid amplification reagent composition of the present invention (1) Sample preparation RNA and DNA were extracted from HeLa cells, respectively. The nucleic acid was electrophoresed on a 1% agarose gel and stained with ethidium bromide, and then fluorescence under ultraviolet irradiation was detected. The number of cells used for nucleic acid extraction and the amount of nucleic acid used for electrophoresis at this time are shown in Table 1, and a photograph obtained by the electrophoresis is shown in FIG. The lane numbers in FIG. 1 correspond to the sample numbers in Table 1. The electrophoresis was performed at a constant voltage of 100 V for 30 minutes. In addition to the reaction solution, a molecular weight marker was also run at the same time, which was used as a reference when comparing the lengths of the detected DNA fragments. The procedure as well as other conditions followed the technique described in Maniatis et al. Molecular Cloning (1982).
（２）試料に含まれるリボ核酸の増幅
表１におけるサンプル番号１〜４の核酸について、それぞれ電気泳動に用いた量を試料としてＧ３ＰＤＨ遺伝子を標的リボ核酸とする増幅反応を行った。反応液組成は、ＤＮａｓｅI（ロシュ製）５００単位／ｍｌ、Ｍ−ＭＬＶ由来逆転写酵素ＲＮａｓｅＨ活性除去変異体（東洋紡績製）５００単位／ｍｌ、熱安定性ＤＮＡポリメラーゼＫＯＤ Ｄａｓｈ（東洋紡績製）２５単位／ｍｌ、ＲＮアーゼ阻害剤（東洋紡績製）５００単位／ｍｌ、Ｇ３ＰＤＨプライマーセット（東洋紡績製）各２００ｎＭ、ｄＡＴＰおよびｄＧＴＰおよびｄＣＴＰおよびｄＴＴＰを各０．４ｍＭ、および１倍濃度の増幅用緩衝液（東洋紡績製、室温下で測定した場合のｐＨが８．６のトリス緩衝液および３．７ｍＭのＭｇを含む）である。 また、Mnイオンの添加は行われていない。ただし、本検討におけるＤＮａｓｅIおよびＭ−ＭＬＶ由来逆転写酵素ＲＮａｓｅＨ活性除去変異体の有効性を確認する目的で、表２に示すように一部の酵素を添加しない反応液も用意した。実際の使用にあたっては、反応液に試料溶液を添加し、反応液量を５０μｌとして、以下の反応に供した。反応条件は以下の通りである：
(2) Amplification of ribonucleic acid contained in the sample The nucleic acids of Sample Nos. 1 to 4 in Table 1 were subjected to an amplification reaction using the G3PDH gene as a target ribonucleic acid with the amount used for electrophoresis as the sample. The reaction solution composition was DNase I (Roche) 500 units / ml, M-MLV-derived reverse transcriptase RNase H activity-removed mutant (Toyobo) 500 units / ml, thermostable DNA polymerase KOD Dash (Toyobo) 25 Unit / ml, RNase inhibitor (manufactured by Toyobo) 500 units / ml, G3PDH primer set (manufactured by Toyobo) 200 nM each, dATP and dGTP and dCTP and dTTP each 0.4 mM, and 1 × concentration buffer for amplification A liquid (manufactured by Toyobo Co., Ltd., containing a Tris buffer solution having a pH of 8.6 when measured at room temperature and 3.7 mM Mg). Further, Mn ions are not added. However, for the purpose of confirming the effectiveness of DNase I and M-MLV-derived reverse transcriptase RNase H activity-removed mutants in this study, reaction solutions to which some enzymes were not added as shown in Table 2 were also prepared. In actual use, the sample solution was added to the reaction solution to make the reaction solution volume 50 μl and subjected to the following reaction. The reaction conditions are as follows:
ＤＮＡ分解反応 ４℃、１５分
逆転写反応 ４２℃、２０分
ＰＣＲ反応 熱変性 ：９８℃、１０秒
アニーリング：６０℃、２秒
伸長反応 ：７４℃、３０秒
DNA degradation reaction 4 ° C, 15 minutes Reverse transcription reaction 42 ° C, 20 minutes PCR reaction Thermal denaturation: 98 ° C, 10 seconds
Annealing: 60 ° C, 2 seconds
Elongation reaction: 74 ° C, 30 seconds
上記ＤＮＡ分解反応および逆転写反応は１回のみ、ＰＣＲ反応（熱変性、アニーリング、伸長反応）は３５回繰り返した。これらの操作はパーキンエルマー社のＤＮＡサーマルサイクラー（ＧｅｎｅＡｍｐ９７００）を用いて行った。増幅反応後の反応液５μｌを２％アガロースゲルにて電気泳動し、エチジウムブロマイド染色した後、紫外線照射下での蛍光を検出した。アガロース電気泳動で得られた写真を図２に示す。なお、図２のレーン番号は表２のサンプル番号に対応する。電気泳動の条件は、定電圧１００Ｖ、３０分間にて行った。反応液の他に分子量マーカーも同時に泳動し、検出されたＤＮＡ断片の鎖長を比較する際の参考とした。 The DNA degradation reaction and reverse transcription reaction were repeated only once, and the PCR reaction (thermal denaturation, annealing, extension reaction) was repeated 35 times. These operations were carried out using a Perkin Elmer DNA thermal cycler (GeneAmp 9700). After the amplification reaction, 5 μl of the reaction solution was electrophoresed on a 2% agarose gel and stained with ethidium bromide, and then fluorescence under ultraviolet irradiation was detected. A photograph obtained by agarose electrophoresis is shown in FIG. The lane numbers in FIG. 2 correspond to the sample numbers in Table 2. The electrophoresis was performed at a constant voltage of 100 V for 30 minutes. In addition to the reaction solution, a molecular weight marker was also run at the same time, which was used as a reference when comparing the lengths of the detected DNA fragments.
（３）結果
図１（および表１）のサンプル番号１〜３より明らかなように、細胞試料によっては、抽出したリボ核酸（ＲＮＡ）に大量のデオキシリボ核酸（ＤＮＡ）が混入することがある。図１のサンプル番号４に対照として電気泳動した抽出ＤＮＡと比較すると、例えば図１のサンプル番号２の抽出ＲＮＡに混入しているＤＮＡは試料とした細胞の１０％前後に相当する量に及ぶ。ＤＮＡとＲＮＡは化学的に極めて類似した物質であるため、このように特に両核酸を含有する生体細胞などを試料とする場合では、抽出ＲＮＡからＤＮＡを完全に排除することは極めて困難である。同様に図１のサンプル番号４に電気泳動した抽出ＤＮＡにも、電気泳動で検出できない量のＲＮＡが混入していることが予想される。
(3) Results As is clear from sample numbers 1 to 3 in FIG. 1 (and Table 1), depending on the cell sample, a large amount of deoxyribonucleic acid (DNA) may be mixed into the extracted ribonucleic acid (RNA). Compared with the extracted DNA electrophoresed on sample No. 4 in FIG. 1 as a control, for example, the DNA mixed in the extracted RNA of sample No. 2 in FIG. 1 covers an amount corresponding to about 10% of the sample cells. Since DNA and RNA are chemically very similar substances, it is extremely difficult to completely exclude DNA from extracted RNA, particularly when a biological cell or the like containing both nucleic acids is used as a sample. Similarly, it is expected that the extracted DNA electrophoresed on sample number 4 in FIG. 1 is also contaminated with an amount of RNA that cannot be detected by electrophoresis.
図２（および表２）より明らかなように、本発明の試薬組成（サンプル番号１〜４）では約４５０ｂｐの部分に明瞭な増幅を確認できるが、ＲＮＡ増幅試薬組成に逆転写酵素が含まれない、すなわち本発明のリボ核酸増幅試薬組成から逆転写活性を有する酵素を除去した場合（サンプル番号５〜８）には増幅を確認できない。したがって、図２のサンプル番号１〜４で確認された増幅は各試料に含まれるＲＮＡに起因する。一方、ＲＮＡ増幅試薬組成にＤＮＡ分解酵素が含まれない、すなわち本発明のリボ核酸増幅試薬組成からＤＮＡ分解活性を有する酵素を除去した場合では、該組成に逆転写酵素が含まれる場合（サンプル番号９〜１２）および逆転写酵素が含まれない場合（サンプル番号１３〜１６）ともに約４５０ｂｐの部分に明瞭な増幅を確認できる。すなわち、図２のサンプル番号１３〜１６で確認された増幅は各試料に含まれるＤＮＡに起因する偽陽性である。このように本発明のリボ核酸増幅試薬組成ではＤＮＡ分解活性を有する酵素を含んでいることにより、試料に含まれるＲＮＡのみに起因する増幅を確認することが可能である。 As is clear from FIG. 2 (and Table 2), in the reagent composition of the present invention (sample numbers 1 to 4), clear amplification can be confirmed at a portion of about 450 bp, but reverse transcriptase is included in the RNA amplification reagent composition. In other words, when the enzyme having reverse transcription activity is removed from the ribonucleic acid amplification reagent composition of the present invention (sample numbers 5 to 8), amplification cannot be confirmed. Therefore, the amplification confirmed in sample numbers 1 to 4 in FIG. 2 is attributed to RNA contained in each sample. On the other hand, when the RNA amplification reagent composition does not contain a DNA degrading enzyme, that is, when an enzyme having DNA degrading activity is removed from the ribonucleic acid amplification reagent composition of the present invention, the composition contains a reverse transcriptase (sample number). 9-12) and when no reverse transcriptase is contained (sample numbers 13 to 16), clear amplification can be confirmed at a portion of about 450 bp. That is, the amplification confirmed in sample numbers 13 to 16 in FIG. 2 is a false positive attributed to the DNA contained in each sample. As described above, since the ribonucleic acid amplification reagent composition of the present invention contains an enzyme having DNA degrading activity, it is possible to confirm amplification caused only by RNA contained in the sample.
実施例２
既存のリボ核酸増幅試薬組成との増幅効率の比較
（１）試料の調製
実施例１においてＨｅＬａ細胞より抽出したＲＮＡ溶液（図１におけるサンプル番号１）を１０倍ずつ希釈した段階希釈液を調製し、試料とした。
Example 2
Comparison of amplification efficiency with existing ribonucleic acid amplification reagent composition (1) Preparation of sample A serial dilution was prepared by diluting the RNA solution extracted from HeLa cells in Example 1 (sample number 1 in FIG. 1) by 10 times. A sample was prepared.
（２）試料に含まれるリボ核酸の増幅
I．本発明のリボ核酸増幅試薬組成による増幅
実施例１と同様の方法により、表３に示す試料についてそれぞれＧ３ＰＤＨ遺伝子を標的リボ核酸とする増幅反応を行った。
(2) Amplification of ribonucleic acid contained in the sample
I. Amplification using the ribonucleic acid amplification reagent composition of the present invention By the same method as in Example 1, each sample shown in Table 3 was subjected to an amplification reaction using the G3PDH gene as a target ribonucleic acid.
II．従来のリボ核酸増幅試薬組成による増幅
表３に示す試料にそれぞれＤＮａｓｅI（ロシュ製）５００単位／ｍｌを添加し、４℃で１５分間反応させ、試料に含まれるＤＮＡを分解した。反応後の溶液を９９℃で１０分間加熱してＤＮａｓｅIを失活させた後、該溶液にＭ−ＭＬＶ由来逆転写酵素ＲＮａｓｅＨ活性除去変異体（東洋紡績製）５００単位／ｍｌ、ＲＮアーゼ阻害剤（東洋紡績製）５００単位／ｍｌ、Ｇ３ＰＤＨプライマーセット（東洋紡績製）各２００ｎＭ、ｄＡＴＰおよびｄＧＴＰおよびｄＣＴＰおよびｄＴＴＰを各０．４ｍＭ、および１倍濃度の増幅用緩衝液（東洋紡績製）を添加し、４２℃で２０分間反応させ、逆転写反応を行った。反応後の溶液を９９℃で１０分間加熱して逆転写酵素を失活させた後、熱安定性ＤＮＡポリメラーゼＫＯＤ Ｄａｓｈ（東洋紡績製）２５単位／ｍｌを添加し、増幅反応を行った。増幅反応の条件は以下の通りである：
II. Amplification by Conventional Ribonucleic Acid Amplification Reagent Composition DNase I (Roche) 500 units / ml was added to each sample shown in Table 3 and reacted at 4 ° C. for 15 minutes to decompose the DNA contained in the sample. The solution after the reaction was heated at 99 ° C. for 10 minutes to inactivate DNaseI, and then M-MLV-derived reverse transcriptase RNaseH activity-removed mutant (manufactured by Toyobo) 500 units / ml, RNase inhibitor (Toyobo Co., Ltd.) 500 units / ml, G3PDH primer set (Toyobo Co., Ltd.) 200 nM each, dATP and dGTP and dCTP and dTTP 0.4 mM each, and 1-fold concentration amplification buffer (Toyobo Co., Ltd.) added The reaction was allowed to proceed at 42 ° C. for 20 minutes to carry out a reverse transcription reaction. The solution after the reaction was heated at 99 ° C. for 10 minutes to inactivate the reverse transcriptase. Then, 25 units / ml of thermostable DNA polymerase KOD Dash (manufactured by Toyobo) was added to carry out an amplification reaction. The conditions for the amplification reaction are as follows:
熱変性： ９８℃、１０秒
アニーリング：６０℃、２秒
伸長反応： ７４℃、３０秒
Thermal denaturation: 98 ° C., 10 seconds Annealing: 60 ° C., 2 seconds Extension reaction: 74 ° C., 30 seconds
上記熱変性、アニーリング、伸長反応は３５回繰り返した。これらの操作はパーキンエルマー社のＤＮＡサーマルサイクラー（ＧｅｎｅＡｍｐ９７００）を用いて行った。 The heat denaturation, annealing, and extension reaction were repeated 35 times. These operations were carried out using a Perkin Elmer DNA thermal cycler (GeneAmp 9700).
I．本発明のリボ核酸増幅試薬組成による増幅、II．従来のリボ核酸増幅試薬組成による増幅ともに、増幅反応後の反応液５μｌを２％アガロースゲルにて電気泳動し、エチジウムブロマイド染色した後、紫外線照射下での蛍光を検出した。アガロース電気泳動で得られた写真を図３に示す。なお、図３のレーン番号は表３のサンプル番号に対応する。電気泳動の条件は、定電圧１００Ｖ、３０分間にて行った。反応液の他に分子量マーカーも同時に泳動し、検出されたＤＮＡ断片の鎖長を比較する際の参考とした。 I. Amplification by the ribonucleic acid amplification reagent composition of the present invention, II. In the amplification using the conventional ribonucleic acid amplification reagent composition, 5 μl of the reaction solution after the amplification reaction was electrophoresed on a 2% agarose gel and stained with ethidium bromide, and then fluorescence under ultraviolet irradiation was detected. A photograph obtained by agarose electrophoresis is shown in FIG. The lane numbers in FIG. 3 correspond to the sample numbers in Table 3. The electrophoresis was performed at a constant voltage of 100 V for 30 minutes. In addition to the reaction solution, a molecular weight marker was also run at the same time, which was used as a reference when comparing the lengths of the detected DNA fragments.
（３）結果
図３（および表３）のサンプル番号１〜３より明らかなように、本発明のリボ核酸増幅試薬組成によって１工程でリボ核酸を増幅させた場合（サンプル番号１〜７）および従来のリボ核酸増幅試薬組成によって３工程でリボ核酸を増幅させた場合（サンプル番号８〜１４）について、増幅効率は同様であった。すなわち、本発明のリボ核酸増幅試薬組成では従来の方法と同様の増幅効率を実現しながら、増幅に必要な作業工程数を大幅に短縮することが可能である。また、該工程数の短縮は単に手間が軽減されるだけでなく、研究や診断の現場において大きな問題となっている、増幅反応の途中で容器の蓋を開閉することによるコンタミネーションの予防策としても重要な効果である。
(3) Results As is clear from sample numbers 1 to 3 in FIG. 3 (and Table 3), when ribonucleic acid was amplified in one step with the ribonucleic acid amplification reagent composition of the present invention (sample numbers 1 to 7) and When the ribonucleic acid was amplified in 3 steps by the conventional ribonucleic acid amplification reagent composition (sample numbers 8 to 14), the amplification efficiency was the same. That is, with the ribonucleic acid amplification reagent composition of the present invention, the number of work steps necessary for amplification can be greatly shortened while realizing the same amplification efficiency as in the conventional method. Moreover, shortening the number of steps not only reduces labor, but is a major problem in research and diagnosis, and as a preventive measure against contamination by opening and closing the container lid during the amplification reaction. Is also an important effect.
本発明により、試料に含まれるデオキシリボ核酸の分解（偽陽性予防処理）とリボ核酸の増幅を１工程で行うことができ、特に研究や診断の現場において人的または時間的な効率を大きく向上させることが可能となる。また、本発明の波及効果として、該現場において大きな問題となっている、増幅反応の途中で容器の蓋を開閉することによるコンタミネーションの予防策を提供することができる。 According to the present invention, deoxyribonucleic acid contained in a sample can be decomposed (false positive prevention treatment) and ribonucleic acid can be amplified in one step, which greatly improves human or temporal efficiency particularly in the field of research and diagnosis. It becomes possible. Further, as a ripple effect of the present invention, it is possible to provide a preventive measure against contamination by opening and closing the lid of the container during the amplification reaction, which is a major problem in the field.
Claims (9)
（ａ）ＤＮＡエンドヌクレアーゼ活性を有する酵素により試料に含まれるＤＮＡを分解し、
（ｂ）逆転写活性を有する酵素により試料に含まれるＲＮＡと相補的なＤＮＡ鎖を合成し、
（ｃ）ＤＮＡポリメラーゼ活性を有する酵素により標的ＲＮＡと相補的なＤＮＡを増幅するための成分を含有する試薬。 An RT-PCR reagent characterized by performing RT-PCR in one step using at least two types of enzymes,
(A) degrading DNA contained in a sample with an enzyme having DNA endonuclease activity;
(B) synthesizing a DNA strand complementary to RNA contained in the sample by an enzyme having reverse transcription activity;
(C) A reagent containing a component for amplifying DNA complementary to the target RNA by an enzyme having DNA polymerase activity.
（ｄ）ＤＮＡエンドヌクレアーゼ活性を有する酵素により試料に含まれるＤＮＡを分解し、
（ｅ）逆転写活性を有する酵素により試料に含まれるＲＮＡと相補的なＤＮＡ鎖を合成し、
（ｆ）ＤＮＡポリメラーゼ活性を有する酵素により標的ＲＮＡと相補的なＤＮＡを増幅すること、からなる方法。 An RT-PCR method comprising performing RT-PCR in one step using at least two types of enzymes, wherein the DNA contained in a sample is degraded by an enzyme having a DNA endonuclease activity in the following step (d): ,
(E) synthesizing a DNA strand complementary to RNA contained in the sample by an enzyme having reverse transcription activity;
(F) A method comprising amplifying DNA complementary to a target RNA by an enzyme having DNA polymerase activity.
The method according to any one of claims 5 to 8 , wherein the enzyme having DNA endonuclease activity is DNase I.
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