阅读说明：本技术 cDNA的合成方法、靶RNA的检测方法及试剂盒 (Method for synthesizing cDNA, method for detecting target RNA, and kit ) 是由 增本佳那子 相原祐希 于 2019-05-28 设计创作，主要内容包括：本发明涉及cDNA的合成方法、靶RNA的检测方法及试剂盒。本发明的课题在于,提供可以以更高的精度由靶RNA合成及扩增cDNA的手段。通过使用与靶RNA杂交的第1寡核苷酸分子、与该第1寡核苷酸分子杂交的第2寡核苷酸分子和逆转录酶且以靶RNA为模板来合成cDNA,从而解决了上述课题。(The present invention relates to a method for synthesizing cDNA, a method for detecting target RNA, and a kit. The present invention addresses the problem of providing means that can synthesize and amplify cDNA from target RNA with higher accuracy. The above object is achieved by synthesizing cDNA using a 1 st oligonucleotide molecule hybridized with a target RNA, a 2 nd oligonucleotide molecule hybridized with the 1 st oligonucleotide molecule, and a reverse transcriptase, and using the target RNA as a template.)

1. A method for synthesizing cDNA by mixing a target RNA, a 1 st oligonucleotide molecule, a 2 nd oligonucleotide molecule and a reverse transcriptase and synthesizing cDNA using the target RNA as a template,

The 1 st oligonucleotide molecule has a 1 st region hybridizing with the target RNA at the 3 'end and a 2 nd region hybridizing with the 2 nd oligonucleotide molecule at the 5' side of the 1 st region.

2. The method according to claim 1, wherein the 1 st oligonucleotide molecule hybridizes to the target RNA via the 1 st region and to the 2 nd oligonucleotide molecule via the 2 nd region,

The reverse transcriptase extends the 3' end of the 1 st oligonucleotide molecule hybridized with the target RNA and the 2 nd oligonucleotide molecule to synthesize cDNA.

3. The method of claim 1, wherein the target RNA is a small RNA.

4. The method according to claim 3, wherein the small RNA is any one selected from miRNA, snRNA, snorRNA, piRNA, pri-miRNA, pre-miRNA, siRNA and shRNA.

5. The method according to claim 1, wherein the base length of the 1 st region of the 1 st oligonucleotide molecule is 3 or more and 15 or less.

6. The method according to claim 1, wherein the base length of the 2 nd region of the 1 st oligonucleotide molecule is 10 or more and 40 or less.

7. The method according to claim 1, wherein the base length of the 2 nd oligonucleotide molecule is 35% or more and 100% or less of the base length of the 2 nd region of the 1 st oligonucleotide molecule.

8. The method according to claim 1, wherein the ratio of the number of bases in the 2 nd oligonucleotide molecule that produce a mismatch site when hybridized with the 2 nd region of the 1 st oligonucleotide molecule is within 50%.

9. The method of claim 1, wherein the 2 nd oligonucleotide molecule is modified at its 3' end such that no extension occurs.

10. The method according to claim 1, further comprising a step of amplifying the cDNA.

11. The method according to claim 10, wherein the cDNA amplification step comprises: mixing said cDNA, a forward primer that hybridizes to said cDNA, a reverse primer that hybridizes to the complementary strand of said cDNA and/or a 1 st oligonucleotide molecule, and a polymerase.

12. The method according to claim 10, wherein the cDNA amplification step is performed by real-time PCR.

13. The method according to claim 1, further comprising the steps of: after synthesis of the cDNA, at least a portion of the 2 nd oligonucleotide molecule is removed.

14. The method of claim 1, wherein the 2 nd oligonucleotide molecule comprises uracil.

15. The method according to claim 14, further comprising the steps of: after synthesis of the cDNA, at least a portion of the 2 nd oligonucleotide molecule is cleaved using uracil N glycosylase.

16. The method according to any one of claims 1 to 15, wherein the cDNA synthesis step and the amplification step are performed in the same reaction system.

17. A method for detecting a target RNA, comprising:

Mixing a target RNA, a 1 st oligonucleotide molecule, a 2 nd oligonucleotide molecule and a reverse transcriptase to synthesize a cDNA using the target RNA as a template; and

Amplifying the cDNA and detecting the amplified product; wherein the content of the first and second substances,

The 1 st oligonucleotide molecule has a 1 st region hybridizing with the target RNA at the 3 'end and a 2 nd region hybridizing with the 2 nd oligonucleotide molecule at the 5' side of the 1 st region.

18. The method according to claim 17, wherein in the detection step, the amplification and detection are performed by real-time PCR to quantify the target RNA.

19. A kit comprising a 1 st oligonucleotide molecule that hybridizes to a target RNA, and a 2 nd oligonucleotide molecule that hybridizes to the 1 st oligonucleotide molecule,

The 1 st oligonucleotide molecule has a 1 st region hybridizing with the target RNA at the 3 'end and a 2 nd region hybridizing with the 2 nd oligonucleotide molecule at the 5' side of the 1 st region.

20. the kit of claim 19, further comprising a reverse transcriptase.

21. The kit of claim 19, further comprising dntps.

22. The kit of claim 19, further comprising: a forward primer that hybridizes to a cDNA synthesized by extension of the 1 st oligonucleotide molecule; a reverse primer that hybridizes to the complementary strand of the cDNA.

23. The kit of claim 19, further comprising a polymerase.

24. The kit of claim 19, further comprising a fluorescently labeled probe.

25. An oligonucleotide molecule that is an oligonucleotide molecule that hybridizes to a target RNA,

The oligonucleotide molecule has a 1 st region at the 3 'end, a 2 nd region on the 5' side of the 1 st region,

Said 1 st region is hybridized with said target RNA,

The 2 nd region hybridizes to an oligonucleotide molecule that is different from the oligonucleotide molecule and the target RNA.

Technical Field

The present invention relates to a method for synthesizing cDNA. In addition, the present invention relates to a method for detecting a target RNA. In addition, the invention relates to kits for use in these methods.

Background

In recent years, it has become clear that low-molecular non-coding RNA, typified by small-molecule RNA (mirna), plays an important role in various biological processes such as generation, differentiation, cell proliferation, and apoptosis. The detection and quantification of such functional low-molecular RNA is very important in elucidating life phenomena. Generally, methods commonly used in the detection of RNA are: cDNA is synthesized from RNA by reverse transcription reaction, and the cDNA is amplified and detected by PCR. However, since miRNA has a base length of about 20 bases and is short, it is difficult to design primers that hybridize to both ends of cDNA obtained by reverse transcription.

As a method for synthesizing and amplifying cDNA from miRNA, for example, methods described in patent documents 1 and 2 are known. Patent document 1 discloses: a reverse transcription reaction of miRNA is performed using a primer having a region that does not hybridize to miRNA on the 5' -side, thereby synthesizing cDNA longer than miRNA (see fig. 1 of patent document 1). Further, patent document 2 discloses: a reverse transcription reaction of miRNA is performed using a primer having a stem-loop structure that is a region that hybridizes to miRNA as a protruding portion (see fig. 4 of patent document 2). The method of patent document 2 obtains a cDNA longer than an miRNA by dissociating a stem portion of a primer after a reverse transcription reaction. The methods of patent documents 1 and 2 use the obtained cDNA as a template, thereby facilitating amplification of cDNA by PCR.

disclosure of Invention

Problems to be solved by the invention

The present inventors have found that the methods of patent documents 1 and 2 have room for improvement in the sensitivity and specificity of cDNA synthesis and amplification from low-molecular RNA. The present invention aims to develop a means for synthesizing and amplifying cDNA from a target RNA with higher accuracy.

means for solving the problems

Accordingly, the present invention provides a method of synthesizing cDNA using target RNA as a template by mixing the target RNA, the 1 st oligonucleotide molecule, the 2 nd oligonucleotide molecule and reverse transcriptase. In this method, the 1 st oligonucleotide molecule has a 1 st region hybridizing with the target RNA at the 3 'end and a 2 nd region hybridizing with the 2 nd oligonucleotide molecule on the 5' side of the 1 st region.

The present invention provides a method for detecting a target RNA, comprising: mixing the target RNA, the 1 st oligonucleotide molecule, the 2 nd oligonucleotide molecule and reverse transcriptase to synthesize cDNA using the target RNA as a template; amplifying the cDNA and detecting the amplified product. In this method, the 1 st oligonucleotide molecule has a 1 st region hybridizing with the target RNA at the 3 'end and a 2 nd region hybridizing with the 2 nd oligonucleotide molecule on the 5' side of the 1 st region.

The present invention provides a kit comprising: a 1 st oligonucleotide molecule hybridized to a target RNA, and a 2 nd oligonucleotide molecule hybridized to the 1 st oligonucleotide molecule. In this kit, the 1 st oligonucleotide molecule has a 1 st region hybridizing with the target RNA at the 3 'end and a 2 nd region hybridizing with the 2 nd oligonucleotide molecule on the 5' side of the 1 st region.

[ Effect of the invention ]

According to the present invention, cDNA can be synthesized and amplified from a target RNA with higher accuracy.

Drawings

FIG. 1 is a schematic diagram showing the reaction principle of cDNA synthesis and amplification by the synthesis method of the present embodiment.

Fig. 2A is a diagram showing an example of the external appearance of the reagent cartridge according to the present embodiment.

Fig. 2B is a diagram showing an example of the external appearance of the reagent cartridge according to the present embodiment.

Fig. 2C is a diagram showing an example of the external appearance of the reagent cartridge according to the present embodiment.

Fig. 2D is a diagram showing an example of the external appearance of the reagent cartridge according to the present embodiment.

FIG. 3 is an amplification curve obtained when a cDNA synthesized by the method of patent document 1 is amplified by real-time PCR.

Fig. 4 is an amplification curve when cDNA synthesized by the method of patent document 2 is amplified by real-time PCR.

FIG. 5 is an amplification curve obtained when cDNA is synthesized by the cDNA synthesis method according to the present embodiment by real-time PCR amplification.

FIG. 6A is an amplification curve in real-time PCR amplification of cDNA synthesized by the cDNA synthesis method according to the present embodiment using the 2 nd oligonucleotide molecule having a base sequence completely complementary to the base sequence of the 2 nd region of the 1 st oligonucleotide molecule.

FIG. 6B is an amplification curve in real-time PCR amplification of cDNA synthesized by the cDNA synthesis method according to the present embodiment using the 2 nd oligonucleotide molecule, wherein the 2 nd oligonucleotide molecule contains bases that are not complementary to the base sequence of the 2 nd region of the 1 st oligonucleotide molecule.

FIG. 6C is an amplification curve in real-time PCR amplification of cDNA synthesized by the cDNA synthesis method according to the present embodiment using the 2 nd oligonucleotide molecule, wherein the 2 nd oligonucleotide molecule contains bases that are not complementary to the base sequence of the 2 nd region of the 1 st oligonucleotide molecule.

FIG. 6D is an amplification curve in real-time PCR amplification of cDNA synthesized by the cDNA synthesis method according to the present embodiment using the 2 nd oligonucleotide molecule, wherein the 2 nd oligonucleotide molecule contains bases that are not complementary to the base sequence of the 2 nd region of the 1 st oligonucleotide molecule.

FIG. 6E is an amplification curve in real-time PCR amplification of cDNA synthesized by the cDNA synthesis method according to the present embodiment using the 2 nd oligonucleotide molecule, wherein the 2 nd oligonucleotide molecule contains bases that are not complementary to the base sequence of the 2 nd region of the 1 st oligonucleotide molecule.

FIG. 7A is an amplification curve in real-time PCR amplification of cDNA synthesized by the cDNA synthesis method according to the present embodiment using the 2 nd oligonucleotide molecule introduced with a cap structure at the 3' end.

FIG. 7B is an amplification curve in real-time PCR amplification of cDNA synthesized by the cDNA synthesis method according to the present embodiment using the 2 nd oligonucleotide molecule introduced with a cap structure at the 3' end.

Detailed Description

In the present specification, the expression "hybridization" means: all or part of a given oligonucleotide (or polynucleotide) forms a duplex with all or part of another oligonucleotide (or polynucleotide) under stringent conditions via hydrogen bonding. "stringent conditions" may be conditions that are generally used by those skilled in the art when hybridization of an oligonucleotide (or polynucleotide) is performed. Examples thereof include: conditions under which one oligonucleotide molecule is capable of specifically hybridizing to another oligonucleotide molecule when there is at least 50%, preferably at least 75%, more preferably at least 90% sequence identity between the 2 oligonucleotide molecules. It is known that stringency during hybridization is a function of temperature, salt concentration, base length and GC content of the oligonucleotide, and the concentration of the chaotropic agent contained in the hybridization buffer. As stringent conditions, for example, the conditions described in Sambrook, J. et al, 1998, Molecular Cloning, Laboratory Manual (ed. 2), Cold Spring Harbor Laboratory Press, New York, and the like can be used.

In the present specification, the term "completely complementary base sequences" means: a base sequence of complementary base pairs forming a Watson-Crick model with respect to all bases in all or part of the base sequence of a prescribed oligonucleotide (or polynucleotide). In the present specification, "mismatch site" means: when 2 oligonucleotide molecules (or polynucleotide molecules) are hybridized, a base that is not complementary to a predetermined base in the base sequence of one oligonucleotide molecule (or polynucleotide molecule) is present at a position corresponding to the base sequence of the other oligonucleotide molecule (or polynucleotide molecule), and thus a site that cannot form a complementary base pair of the Watson-Crick model is formed.

In the present specification, "reaction system" means: there are components necessary for the reverse transcription reaction and/or the nucleic acid amplification reaction, and a limited environment in which the reaction occurs. For example, the reaction system of the reverse transcription reaction may be a reaction solution or a fine droplet such as an emulsion contained in a vessel such as a tube, which contains the target RNA, the 1 st oligonucleotide molecule, the 2 nd oligonucleotide molecule, and the reverse transcriptase. "reverse transcriptase" has the same meaning as "RNA-dependent DNA polymerase".

[1. method for synthesizing cDNA ]

In the method for synthesizing a cDNA according to the present embodiment (hereinafter, also referred to as "synthesis method"), a target RNA, the 1 st oligonucleotide molecule, the 2 nd oligonucleotide molecule, and a reverse transcriptase are mixed, and a cDNA is synthesized using the target RNA as a template.

An example of a desired reaction in the synthesis method according to the present embodiment will be described with reference to fig. 1. In this synthesis method, the 1 st oligonucleotide molecule functions as a reverse transcription primer (hereinafter also referred to as "RT primer"). The 1 st oligonucleotide molecule has a 1 st region hybridizing with the target RNA at the 3 'end and a 2 nd region hybridizing with the 2 nd oligonucleotide molecule at the 5' side of the 1 st region. When the target RNA, the 1 st oligonucleotide molecule, the 2 nd oligonucleotide molecule and the reverse transcriptase are mixed, the 1 st oligonucleotide molecule hybridizes to the target RNA through the 1 st region and the 2 nd oligonucleotide molecule hybridizes through the 2 nd region as shown in the left side (1) of FIG. 1. In the example of FIG. 1, the 1 st region has a base sequence completely complementary to the base sequence of the portion including the 3' -end of the target RNA. In this example, the 2 nd oligonucleotide molecule has the same base length as the 2 nd region and has a base sequence completely complementary to the base sequence of the 2 nd region.

The above mixture is subjected to a temperature condition suitable for reverse transcription reaction, whereby the 3' -end of the 1 st oligonucleotide molecule, which has hybridized with the target RNA and the 2 nd oligonucleotide molecule, is extended by the action of reverse transcriptase, as shown in the left side (2) of FIG. 1, thereby synthesizing cDNA. Although the cDNA is strictly the complementary strand portion of the target RNA, the term cDNA in this specification refers to: an oligonucleotide comprising not only the complementary strand of the target RNA but also the 2 nd region. That is, in the present specification, cDNA means: and (3) a 1 st oligonucleotide molecule extended using the target RNA as a template. The synthesized cDNA may be hybridized with the target RNA and/or the 2 nd oligonucleotide molecule, and the target RNA and the 2 nd oligonucleotide molecule may be easily removed by heat denaturation or the like.

As shown in FIG. 1, since the 2 nd region of the 1 st oligonucleotide molecule is a region that does not hybridize to the target RNA, cDNA longer than the target RNA can be obtained. This facilitates the design of primers to be used for the subsequent cDNA amplification. The synthesis method of the present embodiment is not bound by theory or mechanism of action, but it is presumed that: the extension of the 3' -end of the 1 st oligonucleotide molecule in a state where the 2 nd region of the 1 st oligonucleotide molecule and the 2 nd oligonucleotide molecule form a double strand contributes to the improvement of the accuracy and specificity of cDNA synthesis and subsequent cDNA amplification.

In the synthesis method of the present embodiment, the obtained cDNA may be amplified by a DNA amplification method such as a PCR method. In this case, as a template, a mixture after a reverse transcription reaction involving cDNA can be used as it is. In the example of FIG. 1, cDNA is amplified by a conventional PCR method using a primer hybridizing to cDNA (hereinafter referred to as "forward primer"), a primer hybridizing to the complementary strand of the cDNA (hereinafter referred to as "reverse primer"), and polymerase. In cycle 1 of PCR, the forward primer hybridizes to the cDNA as shown in the right side (3) of FIG. 1. The forward primer is then extended by polymerase to synthesize the complementary strand of cDNA.

In cycle 2, the forward primer hybridizes to the cDNA and the reverse primer hybridizes to the complementary strand of the cDNA, as shown in the right side (4) of FIG. 1. Then, each primer is extended by the action of polymerase to obtain a double-stranded DNA. In the case where the mixture after the reverse transcription reaction is directly used for PCR, the reverse primer may be hybridized with the 2 nd oligonucleotide molecule remaining in the mixture in the example of FIG. 1. However, if a sufficiently large amount of reverse primer compared to the 2 nd oligonucleotide molecule is used, there is little effect on the amplification reaction. In the 3 rd and subsequent cycles, amplification products were obtained exponentially using double-stranded DNA as a template, as shown in the right side (5) of FIG. 1.

The following describes each component used in the synthesis method of the present embodiment.

The target RNA is not particularly limited, and may be appropriately selected from any RNA from which cDNA is desired to be synthesized. In this embodiment, in order to prepare the 1 st oligonucleotide molecule as an RT primer, the target RNA is preferably RNA having a known base sequence. Information on the base sequence of RNA can be obtained from databases known in the art, such as GenBank (http:// www.ncbi.nlm.nih.gov/GenBank /), miRBase (http:// www.mirbase.org/search. shtml), and the like.

The synthesis method of the present embodiment is particularly suitable for synthesizing cDNA from small-molecule RNA. Small RNA refers to non-coding RNA with base length less than 200. Examples of the small-molecule RNA include miRNA, small intranuclear RNA (small nuclear RNA), small nucleolar RNA (small nuclear RNA), piwi-interacting RNA (piRNA), primary miRNA (primary miRNA), precursor miRNA (pre-miRNA), short interfering RNA (short interfering RNA) and short hairpin RNA (shRNA). In a preferred embodiment, the target RNA is a single-stranded RNA having a base length of 15 or more and less than 200.

The source of the target RNA is not particularly limited, and may be RNA obtained from a living body or a biological sample, or may be artificially synthesized RNA. The organism is not particularly limited as long as it has a target RNA, and may be a eukaryote or a prokaryote. Preferred organisms are mammals including humans. Examples of the biological sample include organs, tissues, cells, and body fluids collected from a living body. Examples of the body fluid include blood, plasma, serum, lymph, saliva, and urine. The biological sample may be a culture of a microorganism such as a cultured cell or a bacterium, a virus, a culture supernatant, a solubilized product, an extract, or the like. In recent years, it has been known that a large amount of small-molecule RNA such as miRNA is contained in exosome. In the present embodiment, the biological sample may be an exosome isolated from a body fluid or the like of a living body.

Methods for extracting RNA from biological samples are known per se. For example, when the biological sample is a cell or a tissue, the RNA can be extracted as follows. First, a biological sample is mixed with a solubilizing solution containing guanidine thiocyanate and a surfactant. The obtained mixed solution is subjected to physical treatment (stirring, homogenization, ultrasonic disruption, etc.) to release RNA contained in the biological sample into the mixed solution. Thus, RNA can be extracted from a biological sample. The extracted RNA may be purified. For example, RNA can be purified by centrifuging a mixture containing RNA, collecting the supernatant, and subjecting the supernatant to phenol/chloroform extraction. Extraction and purification of RNA in a biological sample can be performed using a commercially available RNA extraction cassette.

The 1 st oligonucleotide molecule is an oligonucleotide molecule having a 1 st region at the 3 'end and a 2 nd region on the 5' side of the 1 st region. As described above, the 1 st oligonucleotide molecule functions as an RT primer in the synthesis method of the present embodiment. The 1 st region is a region that hybridizes to the target RNA, and the 2 nd region is a region that hybridizes to the 2 nd oligonucleotide molecule. In this embodiment, the 1 st oligonucleotide molecule may have a region other than the 1 st region and the 2 nd region as long as it can extend the 3' end by reverse transcription. In a preferred embodiment, the 1 st oligonucleotide molecule consists of the 1 st region and the 2 nd region. The following describes the 1 st and 2 nd regions.

Region 1 is the region comprising the 3' terminus of oligonucleotide molecule 1. The 1 st oligonucleotide molecule functions as an RT primer by hybridizing to a target RNA via the 1 st region and extending the 3' end. The base length of the 1 st region is not particularly limited as long as it can maintain the hybridization with the target RNA during the reverse transcription reaction. The 1 st region has, for example, a base length of 3 to 15, preferably 4 to 12, more preferably 6 to 10. The nucleotide sequence of the 1 st region is not particularly limited as long as it is a nucleotide sequence that can hybridize to a target RNA. Preferably, the base sequence of the 1 st region is a base sequence completely complementary to a part of the base sequence of the target RNA. The portion hybridizing with region 1 in the target RNA is not particularly limited, but is preferably a portion including the 3' -end of the target RNA.

The 2 nd region is a region located on the 5' side of the 1 st region. The 2 nd region may be a region comprising the 5' end of the 1 st oligonucleotide molecule. As described above, the 1 st oligonucleotide molecule hybridizes to the 2 nd oligonucleotide molecule through the 2 nd region. In this embodiment, all of the 2 nd region may be hybridized with the 2 nd oligonucleotide molecule. Alternatively, a portion of the 2 nd region may be hybridized with the 2 nd oligonucleotide molecule. Thus, all or part of region 2 has a base sequence capable of hybridizing to oligonucleotide molecule 2. Preferably, all or part of the base sequence of region 2 is a base sequence that is fully complementary to the entire base sequence of oligonucleotide molecule 2. The base length of the 2 nd region is not particularly limited, but is, for example, 10 to 40 base length, preferably 17 to 40 base length, and more preferably 20 to 40 base length.

In the synthesis method of the present embodiment, the 2 nd region is an additional sequence that increases the length of the complementary strand of the target RNA synthesized by the reverse transcription reaction. Thus, the 2 nd region is preferably a region that hybridizes to the 2 nd oligonucleotide molecule but does not hybridize to the target RNA. The nucleotide sequence of such a 2 nd region can be appropriately determined according to the nucleotide sequence of the target RNA. Since the base sequence of the 2 nd region may affect the efficiency of cDNA amplification to be performed subsequently, it is preferable to determine the base sequence in consideration of Tm value, GC content, and the like.

The 1 st oligonucleotide molecule itself can be prepared by an oligonucleotide synthesis method known in the art. The 1 st oligonucleotide molecule may contain a conventionally known artificial Nucleic Acid such as a Bridge Nucleic Acid (BNA), a Phosphorothioate (PS) -oligomer, a Peptide Nucleic Acid (Peptide Nucleic Acid: PNA), a morpholino oligomer, or a 2 '-O-substituted RNA, as long as it can extend the 3' -end by reverse transcription.

The 2 nd oligonucleotide molecule is an oligonucleotide molecule that hybridizes to all or part of the 2 nd region of the 1 st oligonucleotide molecule. In this embodiment, the 2 nd oligonucleotide molecule preferably does not hybridize to the 1 st region of the 1 st oligonucleotide molecule. The synthetic method of this embodiment is not bound by theory or mechanism of action, but it is presumed that the 2 nd oligonucleotide molecule exerts the following effects: by hybridizing to region 2 of the 1 st oligonucleotide molecule, undesired hybridization and non-specific binding of the polynucleotide on this region 2 is prevented.

The base length of the 2 nd oligonucleotide molecule may be the same as that of the 2 nd region of the 1 st oligonucleotide molecule, or may be shorter than that of the 2 nd region. For example, the 2 nd oligonucleotide molecule has a base length of 35% or more and 100% or less, preferably 35% or more and 95% or less, and more preferably 35% or more and 90% or less of the base length of the 2 nd region of the 1 st oligonucleotide molecule.

When the 2 nd oligonucleotide molecule has a shorter base length than the 2 nd region of the 1 st oligonucleotide molecule, a plurality of 2 nd oligonucleotide molecules, preferably 2 or 3 nd oligonucleotide molecules, may be used. Each of the 2 nd oligonucleotide molecules hybridizes to a different portion of the 2 nd region from each other. The portion of the 2 nd region to which each 2 nd oligonucleotide molecule hybridizes may be adjacent or separate.

The base sequence of the 2 nd oligonucleotide molecule can be determined appropriately according to the base sequence of the 2 nd region of the 1 st oligonucleotide molecule. The base sequence of the 2 nd oligonucleotide molecule may be a base sequence completely complementary to all or part of the 2 nd region of the 1 st oligonucleotide molecule. Alternatively, the base sequence of the 2 nd oligonucleotide molecule may contain a base that generates a mismatch site when hybridizing with the 2 nd region of the 1 st oligonucleotide molecule (hereinafter also referred to as "mismatched base"). For example, the ratio of the number of mismatched bases in the 2 nd oligonucleotide molecule (hereinafter also referred to as "mismatch ratio") is 50% or less, preferably 40% or less, and more preferably 12% or less. The mismatch ratio is calculated by the following equation.

(mismatch rate) { (number of mismatched bases in the 2 nd oligonucleotide molecule)/(base length of the 2 nd oligonucleotide molecule) } × 100

In the case where the 2 nd oligonucleotide molecule contains mismatched bases, the base length of the 2 nd oligonucleotide molecule preferably has a base length of 35% or more, more preferably 100% of the base length of the 2 nd region of the 1 st oligonucleotide molecule.

The 2 nd oligonucleotide molecule may comprise uracil in the base sequence. When the synthesis method of the present embodiment is performed using the 2 nd oligonucleotide molecule containing uracil, part or all of the 2 nd oligonucleotide molecule hybridized with the synthesized cDNA may be decomposed and removed by uracil DNA glycosylase (UNG).

The 2 nd oligonucleotide molecule itself can be prepared by an oligonucleotide synthesis method known in the art. The 2 nd oligonucleotide molecule may comprise a conventionally known artificial nucleic acid such as BNA, PS-oligomer, PNA, morpholino oligomer, 2' -O-substituted RNA, etc.

In this embodiment, the 3' end of the 2 nd oligonucleotide molecule may be modified in such a way that no extension is performed. Examples of such modification include phosphorylation and biotinylation. Further, a modification group such as dideoxyribonucleotide or an amino linker may be introduced into the 3 '-end of the 2 nd oligonucleotide molecule so that the 3' -end is not extended.

In this embodiment, the 2 nd oligonucleotide molecule may be modified such that the binding stability to the 2 nd region is improved. Such modification includes, for example, introduction of a cap structure into the 5 'end or the 3' end. Examples of the cap structure introduced into the 5' -end include pyrenyl and trimethoxystilbenyl. Examples of the cap structure to be introduced into the 3' -end include 2' - (anthraquinone-2-yl-carboxamide) -2' -deoxyuridine (hereinafter, also referred to as "Uaq"). It is known that the introduction of a cap structure increases the Tm value, and thus the binding to the complementary strand is stable.

The reverse transcriptase is not particularly limited, and may be appropriately selected from known reverse transcriptases. Examples of the reverse transcriptase include Avian Myeloblastosis Virus (AMV) reverse transcriptase and Moloney Murine Leukemia Virus (MMLV) reverse transcriptase. In addition, an enzyme contained in a commercially available reverse transcription reaction cassette can be used.

In this embodiment, for example, the target RNA, the 1 st oligonucleotide molecule, the 2 nd oligonucleotide molecule, and the reverse transcriptase are mixed in water (preferably nuclease-free water), and the resulting mixture (hereinafter also referred to as "composition for RT") is subjected to temperature conditions suitable for the reverse transcription reaction to synthesize cDNA. The RT composition may further contain reagents used in a normal reverse transcription reaction, such as a buffer (e.g., Tris-HCl), dNTP (a mixture of dATP, dCTP, dGTP and dTTP), an inorganic salt (e.g., NaCl, KCl, etc.), and an RNase inhibitor. Such reagents are also included in commercially available reverse transcription reaction cassettes. The order of mixing the above components is not particularly limited. In this embodiment, the 1 st oligonucleotide molecule and the 2 nd oligonucleotide molecule may be mixed in advance and used. However, it is not necessary to hybridize the 1 st oligonucleotide molecule to the 2 nd oligonucleotide molecule in advance. These oligonucleotide molecules will hybridize spontaneously under temperature conditions suitable for reverse transcription reactions.

The amount of the 1 st oligonucleotide molecule and the 2 nd oligonucleotide molecule added is not particularly limited. The final concentrations of the 1 st and 2 nd oligonucleotide molecules in the composition for RT can be determined as appropriate within the range of 10nM to 1000nM, preferably 10nM to 250nM, respectively. The final concentration of the 2 nd oligonucleotide molecule in the RT composition may be the same as or different from the final concentration of the 1 st oligonucleotide molecule.

The temperature conditions in the synthesis method of the present embodiment are not particularly changed from those in the ordinary reverse transcription reaction. The temperature conditions for the known reverse transcription reaction can be appropriately selected depending on the type of reverse transcriptase used and the like. In this embodiment, the reverse transcription reaction can be performed using a commercially available thermal cycler.

The cDNA synthesized may be hybridized with the 2 nd oligonucleotide molecule. The synthesis method of the present embodiment may include a step of removing at least a part of the 2 nd oligonucleotide molecule after synthesizing a cDNA, if necessary. For example, when the RT composition after the reverse transcription reaction is heated at 90 to 99 ℃, the 2 nd oligonucleotide molecule is dissociated from the cDNA by thermal denaturation. Where the 2 nd oligonucleotide molecule comprises uracil, at least a portion of the 2 nd oligonucleotide molecule can be cleaved using UNG after cDNA synthesis. For example, UNG is added to the composition for RT after the reverse transcription reaction and incubated at a predetermined temperature (e.g., 25 ℃) to thereby remove uracil from the 2 nd oligonucleotide molecule hybridized to cDNA. Since the portion from which uracil is lost is structurally unstable, the 2 nd oligonucleotide molecule is cleaved at the portion from which uracil is lost when the RT composition is heated at 90 ℃ to 99 ℃. Thereby, part or all of the 2 nd oligonucleotide molecule is decomposed.

In this embodiment, cDNA can be synthesized using 2 or more types of target RNAs having different nucleotide sequences as templates in the same reaction system. In this case, multiple reverse transcription reactions can be performed by using a plurality of types of 1 st oligonucleotide molecules having 1 st regions corresponding to the base sequences of the target RNAs. The base sequences of the 2 nd region may be the same or different from each other among the plurality of 1 st oligonucleotide molecules. The 2 nd oligonucleotide molecule can be appropriately designed based on the base sequence of the 2 nd region of the 1 st oligonucleotide molecule.

The synthesis method of the present embodiment may further include a step of amplifying the cDNA. The cDNA amplification itself can be carried out by a DNA amplification method known in the art, such as a PCR method or a real-time PCR method. For example, cDNA amplification is performed by mixing cDNA, a forward primer that hybridizes to the cDNA, a reverse primer that hybridizes to the complementary strand of the cDNA, and polymerase. The 1 st oligonucleotide molecule is also capable of hybridizing to the complementary strand of the cDNA, and thus the 1 st oligonucleotide molecule may be used in place of the reverse primer in order to amplify the cDNA.

The forward primer and the reverse primer can be designed appropriately based on the nucleotide sequence of the cDNA, that is, the nucleotide sequences of the target RNA and the 2 nd region of the 1 st oligonucleotide molecule. The base length of each primer is usually 5 or more and 50 or less, preferably 10 or more and 40 or less. The primer itself can be prepared by an oligonucleotide synthesis method known in the art.

The amounts of the forward primer and the reverse primer added are not particularly limited. The final concentrations of the forward primer and the reverse primer in the PCR composition can be appropriately determined within the range of 0.1. mu.M to 1.5. mu.M, preferably 0.35. mu.M to 1.5. mu.M, respectively. The final concentration of the reverse primer and the final concentration of the forward primer in the PCR composition may be the same or different.

The forward primer and the reverse primer may contain conventionally known artificial nucleic acids such as BNA, PS-oligomer, PNA, morpholino oligomer, and 2' -O-substituted RNA, and the forward primer and the reverse primer may be labeled with known labeling substances, and examples of the labeling substance include radioisotopes (e.g., ³² P, ³⁵ S, ³ H, ¹⁴ C), fluorescent pigments (e.g., FITC, Texas Red (trademark), Alexa Fluor (trademark), 6-FAM, and TAMRA), biotin, and digoxigenin.

The polymerase is not particularly limited as long as it is a polymerase suitable for DNA amplification. Such a polymerase can be appropriately selected from known thermostable DNA polymerases, and examples thereof include Taq, Pfu, Tth, and KOD. In addition, a polymerase contained in a commercially available PCR kit may be used.

In this embodiment, a double-stranded DNA is obtained using cDNA as a template by mixing cDNA, a forward primer, a reverse primer and/or a 1 st oligonucleotide molecule, and a polymerase in water and subjecting the resulting mixture (hereinafter also referred to as "composition for PCR") to a temperature condition suitable for DNA amplification. The PCR composition may further contain reagents used in ordinary DNA amplification reactions, such as a buffer (e.g., Tris-HCl), dNTPs, and inorganic salts (e.g., NaCl and KCl). Such reagents are also included in commercially available PCR kits and the like. The order of mixing the above components is not particularly limited. In this embodiment, the forward primer and the reverse primer and/or the 1 st oligonucleotide molecule may be mixed in advance and used.

When amplifying cDNA by the real-time PCR method, an intercalator (e.g., SYBR (registered trademark) Green) that emits fluorescence, and a fluorescent-labeled probe (e.g., TaqMan (trademark) probe) that is modified with a fluorescent substance at the 5 '-end and a quencher at the 3' -end may be further added to the PCR composition.

The temperature conditions for amplification of cDNA are not particularly changed from those of the usual PCR method or real-time PCR method. The temperature can be appropriately selected from temperature conditions of known DNA amplification reactions depending on the type of polymerase used, the Tm value of the primer, and the like. In this embodiment, the amplification of cDNA can be carried out using a commercially available thermal cycler or real-time PCR apparatus.

In this embodiment, the composition for RT after the reverse transcription reaction can be used as it is as a cDNA to be a template. The cDNA in this composition may hybridize with the 2 nd oligonucleotide molecule, but the 2 nd oligonucleotide molecule is dissociated from the cDNA by a thermal denaturation step in DNA amplification. When the 2 nd oligonucleotide molecule contains uracil, a part or all of the 2 nd oligonucleotide molecule hybridized with cDNA can be decomposed and removed by adding UNG to the RT composition after the reverse transcription reaction, incubating at a predetermined temperature (for example, 25 ℃) and then performing DNA amplification.

In this embodiment, the cDNA synthesis step and the cDNA amplification step can be performed in the same reaction system. At this time, for example, the target RNA, the 1 st oligonucleotide molecule, the 2 nd oligonucleotide molecule, the reverse transcriptase, the forward primer, the reverse primer and the polymerase are mixed. The resulting mixture is first subjected to a temperature condition suitable for a reverse transcription reaction to synthesize cDNA. The mixture is then subjected to temperature conditions suitable for amplification of DNA to effect amplification of cDNA. Between the reverse transcription reaction and the amplification reaction of the cDNA, the mixture does not need to be transferred to another vessel. Thus, the reverse transcription reaction and the amplification reaction of cDNA can be performed in the same reaction system. Instead of the reverse transcriptase and the polymerase, an enzyme having both reverse transcription activity and DNA polymerase activity (e.g., Tth) may be used. The mixture may further contain reagents used in ordinary reverse transcription and DNA amplification reactions, such as buffer (e.g., Tris-HCl), dNTPs, inorganic salts (e.g., NaCl, KCl, etc.), and RNase inhibitor.

[2. method for detecting target RNA ]

Also included in the scope of the present invention is a method for detecting a target RNA (hereinafter also referred to as "detection method"). In the detection method of the present embodiment, first, a target RNA, a 1 st oligonucleotide molecule, a 2 nd oligonucleotide molecule and a reverse transcriptase are mixed, and cDNA is synthesized using the target RNA as a template. The details of the components used in the cDNA synthesis (reverse transcription reaction), the conditions of the reverse transcription reaction, and the like are the same as those described above for the synthesis method of the present embodiment.

Then, in the detection method of the embodiment, the synthesized cDNA is amplified, and the amplification product of the cDNA is detected. The details of the components used for amplification of cDNA, the conditions of the amplification reaction, and the like are the same as those described above for the synthesis method of the present embodiment. In the present specification, "detecting" includes: qualitatively determining whether an amplification product exists; quantifying the amplification product; and semi-quantitatively detecting the amount of the amplification product present. Semi-quantitative detection means: the amount of amplification product present is shown in such a stepwise manner as "-", "+" (negative, weakly positive, strongly positive), and the like. For example, in the case where a result that an amplification product is detected is obtained, the result indicates the presence of the target RNA.

The means for detecting the amplification product is not particularly limited, and may be appropriately selected from known methods. For example, the amplification product can be detected using electrophoresis. Specifically, the reaction solution after the amplification reaction may be subjected to electrophoresis on an agarose gel containing ethidium bromide to confirm the presence or absence of the amplification product and the amount of the amplification product in the case where the amplification product is present. In addition, the amplification product may be detected by obtaining optical information such as fluorescence intensity, turbidity, and absorbance from the reaction solution after the amplification reaction. For example, the amplification product can be detected by measuring the fluorescence intensity by an intercalator method using a fluorescent substance capable of binding to double-stranded DNA such as SYBR (trademark) Green.

Alternatively, the amplification product may be detected by detecting fluorescence generated from a fluorescently labeled probe (for example, TaqMan (trademark) probe) in which a fluorescent substance is modified at the 5 '-end and a quencher substance is modified at the 3' -end. Such a probe is designed so as to hybridize to a region different from the region to which the forward primer and/or the reverse primer hybridizes in the amplification product to be detected. The final concentration of the fluorescent labeled probe in the PCR composition is not particularly limited, and can be appropriately determined, for example, from a range of 0.1. mu.M to 5. mu.M, preferably 0.2. mu.M to 0.8. mu.M.

[3. kit ]

Kits are also included within the scope of the invention. The kit can be used for carrying out the synthesis method and the detection method of the present embodiment. The kit of the present embodiment comprises a 1 st oligonucleotide molecule hybridized with a target RNA, and a 2 nd oligonucleotide molecule hybridized with the 1 st oligonucleotide molecule. The details of the 1 st oligonucleotide molecule and the 2 nd oligonucleotide molecule are the same as those described above for the synthesis method of the present embodiment.

The containers containing the 1 st and 2 nd oligonucleotide molecules can be stored in a box and provided to a user. The box may be used to pack all of the containers together, or may be used to store a part of the containers. In this case, additional instructions describing the method of using the 1 st oligonucleotide molecule and the 2 nd oligonucleotide molecule, etc. may be packaged together. Fig. 2A shows an example of the reagent kit according to the present embodiment. In the figure, 10 denotes a kit, 11 denotes a 1 st container containing a 1 st oligonucleotide molecule, 12 denotes a 2 nd container containing a 2 nd oligonucleotide molecule, 13 denotes a package, and 14 denotes an additional instruction manual.

The concentration of the 1 st oligonucleotide molecule in the 1 st vessel may be adjusted to the final concentration described above when used to prepare the composition for RT. Therefore, the concentration of the 1 st oligonucleotide molecule in the container can be appropriately determined by the volume ratio of the total amount of the composition for RT to the added amount of the 1 st oligonucleotide molecule. In this embodiment, the concentration of the 1 st oligonucleotide molecule in the 1 st vessel is, for example, 0.05. mu.M or more and 100. mu.M or less, preferably 0.25. mu.M or more and 10. mu.M or less.

The concentration of the 2 nd oligonucleotide molecule in the 2 nd vessel can be determined in the same manner as the concentration of the 1 st oligonucleotide molecule. In the present embodiment, the concentration of the 2 nd oligonucleotide molecule in the 2 nd container is, for example, 0.05. mu.M or more and 100. mu.M or less, preferably 0.25. mu.M or more and 10. mu.M or less. The concentration of the 2 nd oligonucleotide molecule in the 2 nd vessel may be the same as or different from the concentration of the 1 st oligonucleotide molecule in the 1 st vessel.

The kit of the present embodiment may further comprise a reverse transcriptase. The kit of the present embodiment may further include dntps. The reverse transcriptase itself is known in the art, and can be appropriately selected from enzymes derived from retroviruses such as AMV and MMLV. The dNTP may be a mixture of dATP, dCTP, dGTP and dTTP. FIG. 2B shows an example of a kit further comprising a reverse transcriptase and dNTPs. In the figure, 20 denotes a kit, 21 denotes a 1 st container containing a 1 st oligonucleotide molecule, 22 denotes a 2 nd container containing a 2 nd oligonucleotide molecule, 23 denotes a 3 rd container containing a reverse transcriptase, 24 denotes a 4 th container containing dNTPs, 25 denotes a packaging case, and 26 denotes an additional instruction manual. Although not shown, the kit of the present embodiment may further include a buffer suitable for reverse transcription.

The kit of the present embodiment may further include: a forward primer that hybridizes to a cDNA synthesized by extension of the 1 st oligonucleotide molecule; a reverse primer hybridizing to the complementary strand of the cDNA. The details of the forward primer and the reverse primer are the same as those described above for the synthesis method of the present embodiment. FIG. 2C shows an example of a kit further comprising a forward primer and a reverse primer. In the figure, 30 denotes a kit, 31 denotes a 1 st container containing a 1 st oligonucleotide molecule, 32 denotes a 2 nd container containing a 2 nd oligonucleotide molecule, 33 denotes a 3 rd container containing a reverse transcriptase, 34 denotes a 4 th container containing dNTP, 35 denotes a 5 th container containing a forward primer, 36 denotes a 6 th container containing a reverse primer, 37 denotes a packaging box, and 38 denotes an additional instruction manual. Although not shown, the kit of the present embodiment may further include a buffer suitable for reverse transcription.

The concentration of the forward primer in the 5 th vessel may be adjusted to the final concentration described above when used for preparing a PCR composition. Therefore, the concentration of the forward primer in the container can be appropriately determined according to the volume ratio of the total amount of the PCR composition to the amount of the forward primer added. In the present embodiment, the concentration of the forward primer in the 5 th vessel is, for example, 6. mu.M or more and 100. mu.M or less, preferably 15. mu.M or more and 100. mu.M or less.

The concentration of the reverse primer in the 6 th vessel can be determined in the same manner as the concentration of the forward primer. In the present embodiment, the concentration of the reverse primer in the 6 th vessel is, for example, 3. mu.M or more and 100. mu.M or less, preferably 15. mu.M or more and 100. mu.M or less. The concentration of the reverse primer in vessel 6 may be the same as or different from the concentration of the forward primer in vessel 5.

The kit of the present embodiment may further comprise a polymerase. The kit of the present embodiment may further include a fluorescent-labeled probe. The details of the polymerase and the fluorescent-labeled probe are the same as those described above for the synthesis method and the detection method of the present embodiment. FIG. 2D shows an example of a kit further comprising a polymerase and a fluorescent-labeled probe. In the figure, 40 denotes a kit, 41 denotes a 1 st container in which a 1 st oligonucleotide molecule is contained, 42 denotes a 2 nd container in which a 2 nd oligonucleotide molecule is contained, 43 denotes a 3 rd container in which a reverse transcriptase is contained, 44 denotes a 4 th container in which dNTPs are contained, 45 denotes a 5 th container in which a forward primer is contained, 46 denotes a 6 th container in which a reverse primer is contained, 47 denotes a 7 th container in which a polymerase is contained, 48 denotes an 8 th container in which a fluorescent-labeled probe is contained, 49 denotes a packaging box, and 50 denotes additional instructions. Although not shown, the kit of the present embodiment may further include a buffer suitable for a reverse transcription reaction and a buffer suitable for a DNA amplification reaction.

The concentration of the fluorescent labeled probe in the 8 th vessel may be adjusted to the final concentration when used for preparing the PCR composition. Therefore, the concentration of the fluorescent labeled probe in the vessel can be appropriately determined according to the volume ratio of the total amount of the PCR composition to the amount of the fluorescent labeled probe added. In the present embodiment, the concentration of the fluorescent labeled probe in the 8 th container is, for example, 2. mu.M or more and 25. mu.M or less, preferably 2. mu.M or more and 10. mu.M or less.

In this embodiment, at least 2 kinds of the 1 st oligonucleotide, the 2 nd oligonucleotide, the forward primer and the reverse primer may be contained in the same container. The 1 st and 2 nd oligonucleotides are preferably contained in the same container. Alternatively, the forward primer and the reverse primer are contained in the same container. More preferably, the 1 st and 2 nd oligonucleotides are contained in the same container, and the forward and reverse primers are contained in separate containers.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.