JP2012080871A - Method for directly detecting rna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel, simple, quick and practical method for detecting RNAs in a sample.SOLUTION: The method for detecting RNAs in a sample containing organism-derived impurities includes: (1) a step for producing a circularized single-stranded DNA probe by circularizing a single-stranded DNA with complementary sequence in a target RNA; (2) a step for hybridizing the circularized single-stranded DNA probe obtained in the step (1) with RNA in the sample; (3) a step for synthesizing a single-stranded DNA from the RNA-DNA hybrid obtained in the step (2) by a rolling circle amplification (RCA) method with the RNA as a primer; (4) a step for detecting the target RNA by adding a marker to be combined with the target RNA synthesized in the step (3) and detecting the marker; and further, as an option, (5) a step for forming a nick only in the RNA strand of the RNA-DNA hybrid with RNase H after the step (2).

Description

  The present invention relates to a method for directly detecting RNA in a sample, more specifically, a rolling circle amplification method (RNA-Primed Rolling Circle Amplification) in which DNA is amplified by a strand displacement type DNA synthase using RNA to be detected as a primer. A direct detection method of RNA using RPRCA) and its application.

  A culture method is generally used for the purpose of detecting a small number of microorganisms from a complex sample such as food and checking for the presence of living microorganisms present in the sample. However, the detection of microorganisms by the culture method is largely based on a selective medium and requires a culture period of several days, and is not suitable for food poisoning tests or clinical diagnoses that require rapidity. On the other hand, for rapid detection of microorganisms, a method is known in which a unique DNA sequence possessed by a microorganism or the like is detected using a polymerase chain reaction (PCR). However, since DNA is a chemically stable compound, even if a DNA sequence unique to a microorganism or the like is detected by PCR, life and death of the microorganism or the like cannot be determined.

  Since intracellular RNA is rapidly degraded when the cell dies, a method has been developed for detecting living cells such as microorganisms and distinguishing between living and dead by detecting RNA. In particular, an RT-PCR method in which PCR is performed after RNA is converted to DNA by a reverse transcription reaction (RT (reverse transcription) reaction) is well known (for example, Non-Patent Document 1). However, since the PCR method is a reaction system, it requires an expensive device such as a thermal cycler capable of changing the reaction temperature, and the time required for general PCR is at least 2 to 3 hours. Yes. In addition, a real-time detection method capable of performing amplification and detection at the same time is possible. However, such a detection method requires that a thermal cycler has a built-in detection device, which is more expensive than a thermal cycler. Become.

  On the other hand, RT-LAMP method has also been developed in which RNA is rapidly detected after reverse transcription of RNA by LAMP method (Loop-Mediated Isothermal Amplification) using strand displacement type DNA synthase (Non-patent Document 2). . The RT-LAMP method can be detected in about 1 to 2 hours, but the reaction system is complicated and poor in versatility, and requires a machine that can maintain a relatively high temperature (65 ° C.) on the reaction system. It is said. Compared with the PCR method, real-time detection is possible with a low-cost apparatus, but the reaction system is not widely used at present due to the complexity of special primer design.

  Furthermore, in RNA detection by the RT-PCR method or the RT-LAMP method, a so-called carry in which DNA generated until the final amplification reaction is mixed in the next reaction step to induce a non-specific sequential reaction. Cross contamination due to over is always a problem. For this reason, if impurities in the sample, especially genome-derived DNA, are mixed even in a trace amount, false positive results are likely to occur, and in order to make it difficult to detect the target RNA, RNA such as DNA degradation treatment is purified. Requires a process. Accordingly, additional processes and dedicated facilities are required, and more highly trained personnel are required, which tends to increase not only the initial introduction cost but also the running cost. As a result, not only in the livestock industry and food industry, but also in the field of clinical examination, the adoption of a microorganism detection method by nucleic acid amplification tends to be avoided.

  On the other hand, an RNA detection method with a simple reaction apparatus has also been developed, and after passing through the RT reaction, transcription that amplifies RNA again by bacteriophage RNA synthase from the bacteriophage promoter region set in the primer region used in the RT reaction. RNA amplification / detection methods include Transcription-mediated amplification (TMA) (Non-patent Document 3) and NASBA (Nucleic acid sequence-based amplification; NASBA), which combines ribonuclease H (RNase H) with TMA. (Non-Patent Document 4). Since these are reactions at a low temperature (37 ° C.), a special expensive apparatus such as a thermal cycler is unnecessary. However, since the reaction product is RNA, it is necessary to use a special gel electrophoresis apparatus or the like for detection, or to perform RT-PCR after the reaction, and the operation is complicated.

  As mentioned above, since all existing RNA amplification and detection methods are performed via reverse transcription reactions, the time required for the reverse transcription reaction itself even if the experimental operation is simplified or the reaction conditions are optimized. Therefore, it takes about 2 hours or more until detection, and it is difficult to improve the detection speed. Such a problem is a major obstacle in the development of RNA detection methods that require higher speed, for example, the development of detection methods for pathogenic RNA viruses such as pathogenic E. coli O157 and highly pathogenic influenza viruses. It was.

  In recent years, a rolling circle amplification (RCA) method has been studied as a nucleic acid amplification method for solving these problems. In the RCA method, a single-stranded circular DNA probe is hybridized with a DNA primer having a sequence complementary to the DNA probe, and then single-stranded DNA is continuously produced by a strand displacement type DNA synthesis reaction using the DNA probe as a template. Reaction. Therefore, in the RCA method, the presence of a DNA primer or a DNA probe can be determined by detecting single-stranded DNA generated after the DNA synthesis reaction.

  As an RNA detection method, an RNA-Primed Rolling Circle Amplification (RPRCA) method has attracted attention as a new RNA detection method. RPRCA hybridizes a 3 ′ end sequence of a target RNA molecule with a circular DNA probe having a complementary sequence, and then uses the 3 ′ end of the RNA molecule as a starting point for DNA synthesis, and converts single-stranded DNA into RCA. Synthesize continuously by the method. By detecting the single-stranded or double-stranded DNA synthesized in this manner, the target RNA, particularly a messenger RNA (mRNA) derived from a prokaryotic microorganism, or a eukaryotic microRNA (miRNA) without performing a reverse transcription reaction. ) And other small RNA (sRNA) can be detected in a short time.

  So far, as a method for detecting RNA using the RCA method, a method for detecting the RNA by the RCA method using a single-stranded circular DNA probe hybridized with a eukaryotic miRNA has been studied ( Patent Document 1). However, this RCA method has a problem that a signal-to-noise ratio (S / N ratio) at the time of detection is lowered by a DNA by-product synthesized nonspecifically. In order to avoid a decrease in the signal-to-noise ratio due to non-specific DNA by-products, Patent Document 1 prepares circular DNA in advance through a plurality of DNA oligonucleotides, and unreacted single-stranded DNA Although removal is attempted, since it is plural, it becomes easy to form a partial double difference, and an oligonucleotide that forms a double strand cannot be removed by exonuclease alone, so this method is difficult.

  Further, in Patent Document 2, in the cyclization reaction and DNA synthesis, a non-specific double strand formation that causes non-specific DNA synthesis is suppressed by using a thermostable enzyme to raise the reaction temperature. Are considering. However, due to the thermostable enzyme, the signal detection rate is reduced compared to the non-thermostable enzyme.

  In Patent Document 3, a linear padlock DNA probe (Non-patent Document 5) is hybridized with RNA to be detected, and then the padlock DNA probe is circularized to form a single-stranded circular DNA probe. Disclosure. Similarly, in Patent Document 4 and Non-Patent Document 6, after a hybrid of the padlock DNA probe and the RNA to be detected is formed, a circularization reaction of the padlock DNA probe is performed, and further using a detection DNA primer, A method that can verify the presence or absence of RNA to be detected is reported.

  However, when the linear padlock DNA probe is circularized, the linear padlock DNA probe in the reaction solution is not completely circularized, and the unreacted linear padlock DNA probe itself is nonspecific. A double-stranded nucleic acid is formed. In addition, the use of the detection DNA primer further causes a non-specific DNA by-product, which causes a problem that the S / N ratio at the time of detection is greatly reduced.

  A decrease in the S / N ratio derived from non-specific by-products not only lowers the detection sensitivity, but also may cause inaccurate judgments in the detection of the target RNA by producing false positive results. . This inaccurate decision material can cause a reduction in the subject's “Quality Of Life” (QOL), particularly in clinical detection of infectious microorganisms, including viruses.

  Furthermore, in the RPRCA method using a padlock DNA probe, the circularization of the padlock DNA probe on the target RNA by T4 DNA ligase is only about 2.5%, which is significantly less efficient than that occurring on DNA. (Non-Patent Document 7). Even when the ligase used was changed to T4 RNA ligase, the circularization reaction occurred only about 20% of the total, and thus it was revealed that the detection efficiency of RNA with a padlock DNA probe was very poor.

  In addition, since most of the conventional RPRCA methods use the 3 ′ terminal sequence of RNA to be detected as a primer, detectable RNA molecular species are transfer RNA (tRNA), ribosomal RNA (rRNA), and prokaryotes. MRNA, eukaryotic sRNA, or genomic RNA (gRNA) of some RNA viruses. In particular, specific detection of eukaryotic mRNA by the RPRCA method was impossible in principle because a poly A sequence was present at the 3 'end of all mRNAs.

  In Patent Document 4, since 3′-terminal sequences of various RNA species, particularly 3′-terminal sequences of mRNA, are used as primers, a method of removing poly A sequence by ribozyme, or RNA-DNA hybrids are treated with RNase H A method is described in which a nick is introduced and the 3 ′ end of mRNA is allowed to extend as a primer. However, in the same document, only a technique is described, and neither a reaction product actually obtained nor, for example, detected as a band in a gel. Therefore, since the method described in this document does not recognize the problem of the S / N ratio described above, it cannot distinguish between a target product and a non-specific product, and includes a risk of producing a false positive result. It was something to do. Furthermore, the process for preparing a circularized DNA probe in the same document is extremely complicated and cannot be practically used.

  On the other hand, after hybridization of the RNA to be detected and the padlock DNA probe, the padlock DNA probe is circularized by T4 DNA ligase and hybridized by the 3′-5 ′ exoribonuclease activity of the DNA synthase, particularly phi29 DNA polymerase. A method has been developed in which DNA synthesis is initiated using the hybridized RNA portion as a primer after removing the 3 ′ terminal sequence of the RNA to be detected that has not been soyed to the hybridized position (Non-patent Document 8). In addition, RNase III, a double-stranded RNA-specific degrading enzyme, is added to the reaction solution to decompose and remove the RNA portion of the non-hybridized region that may have a secondary structure (double-stranded region). It has been reported that the improvement of the RCA reaction was recognized (Non-patent Document 9).

  However, the 3'-5 'exoribonuclease activity of phi29 DNA polymerase is so weak that it limits the length of RNA that can be removed. Furthermore, since the 3′-5 ′ exoribonuclease activity of phi29 DNA polymerase cannot destroy the secondary structure of RNA and is hardly degraded by RNA molecules that tend to have secondary structure, the RNA region to be hybridized is located on the 3 ′ end side. For example, the 5 ′ EST sequence could not be practically used (Non-Patent Document 10).

  In Non-Patent Document 10, direct detection of RNA is abandoned, and Locked nucleic acid (LNA) is used as a part of the constituent residues of the target RNA-specific DNA primer used in the reverse transcription reaction as an alternative method. Therefore, it has been reported that the detection sensitivity can be increased by increasing the hybridization efficiency to improve the reverse transcription efficiency and performing the RCA reaction with the RNA-specific reverse transcription product to be detected.

  However, this method is effective for eukaryotic mRNAs composed of a plurality of exons, but in yeast and prokaryotic microorganisms composed of one exon, it is strictly distinguished from the remaining genomic DNA. I can't. Furthermore, although it is finally cDNA in Non-Patent Document 10, since DNA is used as a primer, a non-specific DNA by-product is generated. Therefore, since the specificity of amplification / detection is guaranteed only by hybridization of the detection DNA probe complementary to the padlock DNA probe, the complicated operation procedure cannot be discarded.

  Further, in the prior art, it is considered that it is necessary to prevent noise due to foreign substances such as DNA mixed in the RNA sample because of the problem of the low S / N ratio, and in order to detect the target RNA. Furthermore, samples that can be used have been limited such as treating RNA samples with DNase or preparing purified RNA samples (Patent Document 4, Non-Patent Documents 8 and 9).

  As described above, the methods that have been studied so far require an apparatus for managing the temperature cycle of the PCR reaction, or convert the RNA to be detected into DNA by reverse transcription, and do not stop at the equipment surface. There was also a problem of by-products in the amplification reaction due to contamination.

  On the other hand, in the RPRCA method, a detection probe cannot be constructed depending on the type and origin of the RNA to be detected, and there are more RNA molecular species that cannot execute the RPRCA method of a simple procedure. Furthermore, since a reduction in the S / N ratio derived from a non-specific by-product may cause an inaccurate judgment in target RNA detection, it has not reached a practical stage.

  Therefore, as a method for detecting RNA, specific detection is possible if a part of a sequence such as a genomic sequence is known without requiring an expensive device, and it is simpler, quicker, and more sensitive. There was a need to establish a detection method.

US Patent Application Publication No. 2009/0181390 US Patent Application Publication No. 2006/0188893 JP-T-2003-534882 US Patent Application Publication No. 2005/0112639

Selvaratnam S, Schoedel BA, McFarland BL, Kulpa CF.Application of reverse transcriptase PCR for monitoring expression of the catabolic dmpN gene in a phenol-degrading sequencing batch reactor.Appl.Environ.Microbiol. 61: 3981-3985. (1995). Fukuta S, Iida T, Mizukami Y, Ishida A, Ueda J, Kanbe M, Ishimoto Y. Detection of Japanese yam mosaic virus by RT-LAMP. Arch. Virol. 148: 1713-20 (2003). Kwoh DY, Davis GR, Whitfield KM, Chappelle HL, DiMichele LJ, Gingeras TR.Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format.Proc. Natl. Acad. Sci. USA 86: 1173-7 (1989). Guatelli JC, Whitfield KM, Kwoh DY, Barringer KJ, Richman DD, Gingeras TR.Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication.Proc. Natl. Acad. Sci. USA. 87: 1874-1878 (1990). Nilsson M, Malmgren H, Samiotaki M, Kwiatkowski M, Chowdhary B.P., and Landegren U. Padlock probes: circularizing oligonucleotides for localized DNA detection.Science 265 2085-8 (1994).

Wang B, Potter SJ, Lin Y, Cunningham AL, Dwyer DE, Su Y, Ma X, Hou Y, Saksena NK.Rapid and sensitive detection of severe acute respiratory syndrome coronavirus by rolling circle amplification.J. Clin. Microbiol. 43 ( 5): 2339-44 (2005) Li N, Jablonowski C, Jin H, Zhong W. Stand-Alone Rolling Circle Amplification Combined with Capillary Electrophoresis for Specific Detection of Small RNA. Anal. Chem. 81, 4906-4913 (2009) Lagunavicius A, Merkiene E, Kiveryte Z, Savaneviciute A, Zimbaite-Ruskuliene V, Radzvilavicius T, Janulaitis A Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA.RNA 15: 765- 771 (2009). Merkiene E, Gaidamaviciute E, Riauba L, Lagunavicius A. Direct detection of RNA in vitro and in situ by target-primed RCA: the impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 39-end. RNA Published online June 28 (2010) Larsson C, Grundberg I, Soderberg O, Nilsson M. In situ detection and genotyping of individual mRNA molecules. Nat. Methods 7: 395-397 (2010)

  An object of the present invention is an RNA detection method that does not require advanced equipment, can be performed quickly with a simple operation and with practical sensitivity, and includes a sample containing biological contaminants. Among other things, it is to provide a method capable of specific detection even in a sample mixed with DNA.

  As a result of intensive studies to solve the above problems, the present inventors synthesized DNA complementary to the 3 ′ end partial sequence of the target RNA to be detected, and self-circularized with a single-stranded DNA ligating enzyme. When the resulting circularized single-stranded DNA probe is hybridized with the target RNA and single-stranded DNA is synthesized using the target RNA as a primer, a single-stranded DNA-specific fluorescent dye is previously prepared in the reaction system. It has been found that the target RNA can be detected in real time by adding.

  In addition, by adding the RNase H treatment step, it is possible to detect even RNA having no complementary sequence to the DNA probe at the 3 ′ end, and various RNAs derived from eukaryotes as well as viruses and prokaryotes. Can be detected.

  Furthermore, the present inventors have mixed, for example, DNA in a sample containing biological contaminants based on the knowledge that the target RNA was detected in the sample actually obtained from the cells by the RPRCA method of the present invention. It was found that even a sample can detect a specific target RNA, and the present invention has been completed.

That is, the present invention relates to the following methods and kits.
[1] A method for detecting RNA in a sample containing contaminants derived from an organism,
(1) circularizing single-stranded DNA having a sequence complementary to RNA to be detected to produce a circularized single-stranded DNA probe;
(2) a step of hybridizing the circularized single-stranded DNA probe obtained in (1) above with RNA in the sample,
(3) A step of synthesizing single-stranded DNA from the RNA-DNA hybrid obtained in the above (2) by rolling circle amplification (RCA) using RNA as a primer,
(4) The method comprising the steps of adding a label that binds to the single-stranded DNA synthesized in (3) and detecting the RNA to be detected by detecting the label.
[2] The method according to [1], wherein the contaminant derived from an organism contains DNA.
[3] The method according to [1] or [2], wherein the sample is a nucleic acid sample.
[4] The method according to [2] or [3], wherein the DNA in the sample is not decomposed.
[5] The method according to any one of [1] to [4], which comprises decomposing unreacted single-stranded DNA that has not been circularized in (1).
[6] The method according to any one of [1] to [5], wherein the circularized single-stranded DNA probe comprises only a sequence complementary to the RNA to be detected.
[7] The method according to any one of [1] to [6], wherein the circularized single-stranded DNA probe has a length of 30 to 4500 bases.
[8] After (2),
(5) The method according to any one of [1] to [7], further comprising a step of forming a nick only in the RNA strand of the RNA / DNA hybrid by RNase H.
[9] The method according to [8], wherein (3) and / or (5) is performed at a constant temperature in the range of 25 ° C to 65 ° C.
[10] The method according to [8] or [9], wherein in (5), RNase H is used at a final concentration of 1 × 10 ⁻⁴ to 10 units / reaction.
[11] The method according to [8] or [9], wherein, in (5), RNase H is used at a final concentration of 1 × 10 ^{−7 to} 0.1 unit / μl.
[12] The method according to any one of [1] to [11], wherein the RNA is derived from a eukaryote, a prokaryote, a virus, or a viroid.
[13] The method according to [12], wherein the RNA has a poly A sequence.
[14] The method according to [12], wherein the RNA is non-coding RNA.
[15] A method for detecting eukaryotes, prokaryotes, viruses or viroids, comprising the method for detecting RNA according to any one of [1] to [14].
[16] The method according to [15], wherein a microorganism is detected.
[17] The method for detecting RNA according to any one of [1] to [14] and / or the method for detecting eukaryote, prokaryote, virus or viroid according to [15], and / or [ [16] A kit for use in the method for detecting a microorganism according to [16], comprising a circularized single-stranded DNA probe comprising a sequence complementary to the RNA to be detected.
[18] The kit according to [17], further comprising one or more selected from the group consisting of deoxynucleotides consisting of adenine, guanine, cytosine and thymine, a reaction buffer and a strand displacement DNA synthase .

According to the present invention, it is not necessary to perform a reverse transcription reaction with respect to a target RNA to be detected at the time of RNA detection, and DNA is synthesized using RNA directly as a primer, and the synthesized single-stranded DNA By reacting with a single-strand-specific fluorescent dye, real-time detection can be performed very simply.
In addition, non-specific DNA synthesis derived from unreacted single-stranded DNA can be completely suppressed, and a high signal-to-noise ratio (S / N ratio) is realized. The risk of cross-contamination due to overruns is significantly lower than other nucleic acid amplification methods, so RNA can be easily operated with practical sensitivity, and does not require advanced equipment. It becomes possible to detect quickly in time.

  In addition, by adding an RNase H treatment step, various types of RNA can be detected if it is partially known without depending on the sequence of the 3 ′ end of the RNA to be detected. In addition to RNA derived from or prokaryotes, eukaryotic RNA and those organisms themselves can be detected.

  Furthermore, according to the present invention, since the detection sensitivity is good, it is possible to detect RNA even if a sample contains a biological contaminant. Even if the sample contains DNA, it does not require DNA degradation. Therefore, the method of the present invention can detect RNA in a sample quickly and easily as compared with conventional RNA detection methods. This is because even if the contaminating DNA is hybridized with the circularized single-stranded DNA probe, it is almost impossible to use the 3 'end as a primer.

  This is a surprising finding contrary to the common technical knowledge in the art of removing contaminating DNA in a sample in order to specifically detect RNA, and this facilitates detection of RNA derived from living cells. As a result, it is possible to detect a specific RNA that is simple and quick while having sufficient sensitivity for practical use. Therefore, not only research in medicine, pharmacy, agriculture, food science, but also medical fields such as clinical tests, simple viable bacteria tests in the food industry, rapid tests in food poisoning, etc. require simplicity, labor saving, and speed. It is particularly useful in various basic and applied fields.

FIG. 1 is a schematic view showing detection of RNA by RNA-primed RCA of the present invention. FIG. 2 shows specific detection of in vitro transcribed mRNA by RNA-primed RCA: (A) shows the 3 'end sequence of GFP mRNA transcribed in vitro. GFP mRNA was prepared by in vitro transcription of the GFP gene after NotI digestion. The underline and italic letters indicate the stop codon and NotI restriction enzyme site of the GFP gene, respectively. Uppercase and lowercase letters in the sequence indicate DNA and RNA residues, respectively. (B) Real-time detection of in vitro transcribed mRNA by RNA-primed RCA. Fluorescence by RNA-primed RCA is indicated by the following symbols: white circles indicate no mRNA or DNA probe, black circles indicate mRNA alone, white squares indicate P1 (probe represented by SEQ ID NO: 1, Table 1) alone, black Squares are P1 and mRNA, white triangles are P2 (probe represented by SEQ ID NO: 2, Table 1) alone, black triangles are P2 and mRNA. (C) Electrophoretic detection of in vitro transcribed mRNA by RNA-primed RCA. DNA amplified by RNA-primed RCA for 15 hours was run on an agarose gel together with a molecular weight marker (kilobase ladder marker). (D) shows the effect of probe length on RNA-primed RCA. Fluorescence by RNA-primed RCA is indicated by the following symbols, respectively: white circle is no mRNA or DNA probe, black circle is mRNA alone, white square is P1 alone, black square is P1 and mRNA, white triangle is P3 (SEQ ID NO: Probe represented by 3, Table 1) alone, black triangles are P3 and mRNA. FIG. 3 shows the sensitivity of RNA-primed RCA: (A) Effect of concentration of phi29 DNA polymerase in RNA-primed RCA with in vitro transcribed GFP as primer. Enzyme concentrations are indicated by the following symbols: white circle is 0 unit, black circle is 10 units, white square is 20 units, black square is 40 units, white triangle is 60 units, black triangle is 80 units, white diamond is 100 unit. (B) Titration analysis of RNA-primed RCA with serially diluted in vitro transcription GFP. The amounts of GFP mRNA used were 0 pg (white circles), 32 pg (black circles), 320 pg (white squares), 3.2 ng (black squares), and 32 ng (white triangles). FIG. 4 shows specific detection of mRNA present in total RNA isolated from E. coli: (A) GFP expression in E. coli harboring pET-AcGFP. GFP expression with or without self-induction was visualized under blue light. (B) 3 'terminal sequence of GFP mRNA transcribed in E. coli. GFP mRNA was transcribed under the control of the T7 promoter, and such transcription was terminated with a T7 terminator. The underline indicates a sequence complementary to the circularization probe P4 (probe represented by SEQ ID NO: 4, Table 1). (C) Real-time detection of GFP mRNA present in total RNA isolated from E. coli with or without self-induction by RNA-primed RCA. Fluorescence by RNA-primed RCA is indicated by the following symbols: (1) white circles are neither mRNA nor DNA probe, (2) gray circles are total RNA derived from E. coli without induction, (3) black circles are E. coli with induction. (4) white square is P4 alone, (5) gray square is P4 and total RNA derived from E. coli without induction, (6) black square is total RNA derived from E. coli with P4 and induction, (7 ) White triangle is P3 alone, (8) Gray triangle is total RNA derived from E. coli without P3 and induction, (9) Black triangle is total RNA derived from E. coli with P4 and induction. (D) Titration analysis of RNA-primed RCA with serial dilutions of total RNA obtained from induced E. coli. The amounts of total RNA used were 0 ng (white circle), 2 ng (black circle), 10 ng (white square), 20 ng (black square), 40 ng (white triangle) and 80 ng (black triangle).

FIG. 5 is a schematic diagram showing detection of RNA by RNA-primed RCA of the present invention, further comprising the step of forming a nick only in the RNA strand of the RNA / DNA hybrid by RNase H. (A) Since the downstream of the mRNA of the target gene (3 'end) does not form a complementary strand with the circularized DNA probe, the DNA synthesis reaction does not start. (B) After the target gene mRNA and the circularized DNA probe form complementary strands, nicks are introduced by treatment with RNase H, and the RCA reaction starts. FIG. 6 shows the entire sequence of the GFP gene cloned into pET-21d. GFP mRNA was prepared by in vitro transcription using NotI digested pET-AcGFP and T7 RNA polymerase. The underline (bold) indicates the position of the circularized DNA probe (P5) designed to form a complementary strand in the GFP mRNA internal region. Underlined ATG and TGA indicate the start and stop codons, respectively. FIG. 7 shows the effect of RNase H treatment in the detection of in vitro transcribed mRNA by RNA-primed RCA. DNA amplified by RNA-primed RCA for 15 hours was run on an agarose gel together with a molecular weight marker (kilobase ladder marker). Moreover, the result of having investigated the density | concentration dependence of RNase H in RNA-primed RCA reaction is shown.

FIG. 8 shows the detection result of contaminating DNA (pET-AcGFP) in the RNA sample by PCR. Middle DNase (+) and DNase (−) indicate presence and absence of DNase treatment, respectively. Lower (+) and (−) indicate total RNA derived from cells in which expression is induced and cells in which expression is not induced. Each is shown. The PCR product was run on an agarose gel with a molecular weight marker (100 bp ladder). FIG. 9 shows specific detection of a target RNA by the RNA-primed RCA of the present invention in a sample contaminated with contaminating DNA. DNA amplified by RNA-primed RCA for 4 hours was run on an agarose gel. The presence or absence of DNase treatment does not affect the detection result of GFP mRNA in the total RNA sample. N represents a negative control, and (+) and (−) represent total RNA derived from cells in which expression was induced and cells in which expression was not induced, respectively.

The method for detecting RNA of the present invention uses a rolling circle amplification (RCA) method using a strand displacement type DNA synthase and uses an applied technique.
Therefore, first, (1) a single-stranded DNA (ssDNA) having a sequence complementary to the RNA to be detected is circularized to produce a circularized single-stranded DNA probe.
As a sequence complementary to RNA to be detected (target RNA), from the viewpoint of target RNA-specific detection, for example, 20 to 100 bases, particularly 25 to 40 bases, on the 3 ′ end side of the target RNA. Complementary sequences are preferred. When a step of introducing a nick into an RNA strand on a DNA-RNA hybrid by RNase H treatment is included, a sequence complementary to the target RNA to be detected can be arbitrarily set from any position of the RNA. is there.

  The ssDNA that forms the complementary strand is preferably synthesized by a nucleic acid synthesizer if it is 100 bases or less, but if it is 100 bases or more, after amplification of the target DNA by PCR or the like, a single strand is prepared by a known method such as heat denaturation After chain formation, it can also be used in a cyclization reaction. For example, a complementary sequence corresponding to 30 to 4,500 bases, particularly 50 to 700 bases is preferable. The detection sensitivity increases as the probe length increases, and decreases as the probe length decreases. In order to further suppress non-specific reactions, the probe may be composed only of a complementary sequence that can hybridize with the RNA to be detected.

The circularization of single-stranded DNA can be performed by any means. For example, CircLigase (registered trademark), CircLigase II (registered trademark), ssDNA Ligase (Epicentre), ThermoPhage ligase (registered trademark) single-stranded It can be performed using DNA (Prokzyme). The circularization may be performed by intramolecular ligation, or may be performed in a state where a plurality of single-stranded DNAs are arranged in tandem by intermolecular ligation. Preferably, a single-stranded DNA ligase of a kind that does not require a guide sequence in the DNA probe in the circularization reaction is used to prevent incomplete exonuclease treatment due to partial double strand formation.
In order to obtain a high S / N ratio, it is preferable to degrade unreacted unreacted single-stranded DNA, and particularly preferably, exonuclease I (each company), T5 exonuclease (Epicentre), exonuclease Enzymatic degradation with exonuclease capable of degrading single-stranded oligonucleotides such as VI (Epicentre) and RecJ exonuclease (Epicentre) to mononucleotide is preferred. However, exonuclease III cannot be used because the prepared single-stranded circular DNA is used as a substrate.

Next, (2) the circularized single-stranded DNA probe obtained in (1) above is hybridized with RNA in the sample. Those skilled in the art can appropriately set the conditions for such hybridization after examining the combination of the circularized single-stranded DNA probe and the target RNA.
The sample used in the present invention is not particularly limited as long as it contains the target RNA, but it can be used whether the RNA is purified or not purified. In order to avoid degradation of the target RNA, the sample may have a reduced ribonuclease activity. For example, it may be a sample treated with a ribonuclease inhibitor to suppress ribonuclease activity.

Typically, it may contain biologically derived contaminants. Examples of biological contaminants include various DNAs such as genomic DNA, chloroplast DNA, mitochondrial DNA, and plasmid DNA, proteins, lipids, and polysaccharides.
Further, when DNA is contained in the sample, it may be a DNA degrading enzyme such as DNase or other DNA decomposing treatment. In particular, since the present invention does not require a DNA degradation treatment, it can be preferably carried out on a sample containing DNA.

Optionally, a step (5) of forming a nick only on the target RNA strand with RNase H on the target RNA-DNA hybrid obtained in (2) above may be included. RNase H is used in addition to commonly used mesophilic RNase HI (each company) and RNase HII (NEB), as well as Hybridase (registered trademark), Thermostable RNase H (Epicentre), Tth RNase H (Toyobo) Heat resistance can also be achieved by using RNase H. Since RNase H is necessary in the initial stage of the reaction and is not necessary in the middle to late stages of the reaction, the amount required may be very small. The amount (concentration) of RNase H used in the DNA elongation reaction is preferably about 1 × 10 ⁻⁴ to 10 units / reaction at a final concentration, more preferably about 1 × 10 ⁻⁴ to 1 unit / reaction at a final concentration. use.
Alternatively, in the method of the present invention, RNase H is preferably used in the range of 1 × 10 ^{−7 to} 0.1 unit / μl, more preferably 1 × 10 ⁻⁷ to 1 × 10 ⁻² unit / μl. It is done.

  Furthermore, (3) Rolling Circle Amplification (RCA) using RNA as a primer is performed from the RNA-DNA hybrid obtained in (2). RCA includes, for example, phi29 polymerase (each company), Klenow DNA Polymerase (5'-3 ', 3'-5' exo minus) (each company), Sequenase (registered trademark) Version 2.0 T7 DNA Polymerase (USB company), Bsu DNA Medium-temperature strand displacement DNA synthetase such as Polymerase, Large Fragment (NEB), Bst DNA Polymerase (Large Fragment) (each company), Bsm DNA Polymerase, Large Fragment (Fermentas), BcaBEST DNA polymerase (TakaraBio) , Vent DNA polymerase (NEB), Deep Vent DNA polymerase (NEB), DisplaceAce (registered trademark) DNA Polymerase (Epicentre) and the like can also be used.

  The DNA elongation reaction according to the method of the present invention preferably does not require the use of a thermal cycler, and is carried out at a constant temperature in the range of 25 ° C to 65 ° C, for example. The reaction temperature is appropriately set by a normal procedure based on the optimum temperature of the enzyme and the denaturation temperature based on the primer chain length (temperature range in which the primer binds (anneals) / dissociates with the template DNA). Furthermore, it is also carried out at certain relatively low temperatures. For example, when phi29 DNA polymerase is used as the strand displacement type DNA synthase, the reaction is preferably performed at 25 ° C to 42 ° C, more preferably about 30 ° C.

(4) A label that binds to the single-stranded DNA synthesized in (3) above is added, and the RNA to be detected is detected by detecting the label. As such a label, for example, various detectable labels such as a radiation (such as ³² P) label and a fluorescent label can be used. In particular, a detector based on ease of detection, safety, use of LED, and the like. From the viewpoint of simplicity during preparation, a fluorescent label is preferable. Moreover, since single-stranded DNA partially forms a double strand, it can be understood by those skilled in the art that ethidium bromide used for general double-stranded DNA detection can also be used for detection of amplification products. right.

The label may be specific to the detection target sequence or not specific as long as it binds to the synthesized single-stranded DNA. Examples of the label that specifically binds to the sequence include a radiolabeled DNA fragment, an RNA fragment containing a modification such as methylation, a peptide nucleic acid (PNA) fragment, an antibody that binds specifically to the DNA sequence, and metal nanoparticles Is mentioned. Specific DNA, RNA, and PNA sequences that are targets of labeling may be sequences that are complementary to the sequence of the RNA to be detected. For example, sequences that are targets of labeling in circular single-stranded DNA probes It is also possible to incorporate a complementary sequence.
Moreover, even if it is not specific to the sequence, it is possible to use a label that specifically binds to DNA. For example, a fluorescent dye such as SYBR Green II (Invitrogen) can be used.

The detection method of a label | marker can be suitably selected according to the kind of label | marker. For example, when an antibody is used for labeling, detection is performed using a secondary antibody or coupling reaction. When metal nanoparticles are used, changes in physical properties associated with changes in interparticle interactions are measured. Can be detected. Furthermore, it is also possible to detect using chromatography, electrophoresis or the like.
In the method for detecting RNA of the present invention, the RNA to be detected is not particularly limited. For example, the expression of messenger RNA (mRNA) in various cells is detected in real time, or RNA derived from microorganisms or viruses is targeted for detection. It is also possible.

  In one embodiment of the present invention, a kit for RNA detection including a reaction reagent and a composition for the RNA detection method described herein can be provided. The kit of the present invention includes a circularized single-stranded DNA probe, a strand-displacing DNA synthase, an RNase H, a dNTP mixture, a reaction buffer, and a detection, which serve as a template for the RNA detection reaction described herein. Fluorescent dyes and the like can be included.

  The RNA to be detected of the present invention is not particularly limited, and can detect all types of RNA including mRNA, ribosomal RNA (rRNA), and transfer RNA (tRNA). The mRNA to be detected may or may not have a poly A sequence. The RNA may be non-coding RNA including siRNA, miRNA, piRNA, rasiRNA, rRNA, tRNA, and may be genomic RNA such as virus. The shape of the RNA to be detected can be either linear or circular.

  In one embodiment of the RNA detection of the present invention, the RNA to be detected may be prepared or isolated from a sample derived from any biological species. A sample containing such RNA can be a virus, prokaryotic or eukaryotic individual itself, or a part thereof. For example, in vertebrates (including humans), feces, urine, or excrement such as sweat, blood, semen, saliva, gastric fluid, bodily fluids such as bile, and the like. Further, it may be a tissue surgically removed from a living body, or a tissue removed from a living body such as body hair. Further, it may be an RNA-containing preparation prepared from a processed product such as food. Further, it may be an RNA-containing preparation prepared by further fractionating the sample and removing a part thereof.

The present invention also relates to a method for detecting an organism.
In the present invention, organisms include prokaryotes, eukaryotes, viruses, and viroids, and can be detected by detecting RNA derived therefrom. The species that can be detected is not particularly limited as long as it has RNA, and the type of RNA is not limited. For example, organisms such as viruses, bacteria, molds, yeasts, insects, plants and animals can be targeted.

Furthermore, one embodiment of the present invention relates to a method for detecting a microorganism.
In the present invention, the microorganism can be detected by detecting RNA derived therefrom. Microorganisms refer to minute organisms whose structural units cannot be observed with the naked eye, including viruses and multicellular organisms. Examples of detectable microorganisms include bacteria that produce mRNA that does not have a poly A sequence, and viruses or viroids whose genome is RNA. In addition, eukaryotic microorganisms including protozoa and fungi such as mold and yeast can also be targeted.

  EXAMPLES The present invention will be described in detail below by examples, but the present invention is not limited to the examples.

〔Materials and methods〕
1. Circularization of template oligonucleotides The oligonucleotides shown in Table 1 were phosphorylated using 10 units of T4 polynucleotide kinase in 1 × T4 ligase buffer containing 1 mM ATP. The phosphorylated oligonucleotide was circularized using 10 units of CircLigase® ssDNA Ligase (Epicentre) in 1 × CircLigase buffer of 2.5 mM MnCl 2 at 65 ° C. for 1 hour. Non-circularized oligonucleotides were digested with 10 units of exonuclease I at 37 ° C. for 1 hour, followed by treatment at 80 ° C. for 20 minutes to inactivate the enzyme. The circularized oligonucleotide was purified by BioSpin (BioRad), and the concentration was measured using Quant-iT ssDNA BR Assay Kit (Invitrogen) and Qubit fluorometer (Invitrogen). Comparison of the digestibility of cyclized oligonucleotides with exonuclease and that of linear oligonucleotides was analyzed by agarose gel electrophoresis (2.0%, Tris-Borate-EDTA buffer) and cyclized by CircLigase reaction. Confirmed that it was done.

2. Cloning of GFP gene into T7 expression vector pET-21d Simultaneous digestion of 2 μg of pAcGFP1 (Clontech) with 50 units of NcoI and NotI (Takara Bio Inc.), respectively, gave a single green fluorescent protein (GFP) gene. Released. The GFP gene was purified by agarose gel electrophoresis and purified using Wizard® SV Gel and PCR Clean-Up Systems (Promega). The concentration of purified DNA was measured using Quant-iT dsDNA HS Assay Kit (Invitrogen) and Qubit fluorometer (Invitrogen). The purified GFP gene was ligated to a T7 expression vector pET-21d (Novagen) cleaved with NcoI and NotI to prepare a recombinant plasmid pET-AcGFP. After transformation into Escherichia coli DH5α strain, plasmid DNA was purified from the selected colonies, treated with restriction enzymes, and confirmed by agarose gel electrophoresis. The concentration of purified pET-AcGFP was measured using a Quant-iT dsDNA HS Assay Kit and a Qubit fluorometer.

3. Transcription of GFP mRNA in vitro Transcription of GFP mRNA in vitro was carried out using MEGAscript (registered trademark) T7 Kit (Ambion). Linear template DNA obtained by digesting 1 μg of pET-AcGFP with NotI was added to the transcription reaction solution and incubated at 37 ° C. for 2 hours. After removing the template DNA by DNase I treatment, the transcribed mRNA was purified using MEGAclear (registered trademark) Kit (Ambion), and the concentration was measured by spectroscopic analysis. The transcribed mRNA was stored at −80 ° C. until use.

4). In vivo expression of GFP mRNA and RNA extraction Escherichia coli BL21 (DE3) strain (Novagen) was transformed with pET-AcGFP and LB agar plates (50% / ml ampicillin for selection of pET-AcGFP) (1% tryptone) , 0.5% yeast extract, 1.0% NaCl and 1.5% agar). E. coli was cultured in LB medium at 30 ° C. for 8 hours. The culture was diluted 1: 1000 with fresh LB medium containing Overnight Express (registered trademark) Autoinduction System (Novagen), cultured at 30 ° C for 16 hours with shaking, and GFP mRNA was transcribed with T7 RNA polymerase encoded by DE3. Induced. E. coli was recovered in a 50 ml conical tube by centrifugation at 3,500 rpm at 4 ° C. for 20 minutes. Total RNA was isolated using Trizol® Max Bacterial RNA Isolation kit (Invitrogen). The prepared total RNA was used as a sample without performing DNase treatment (for example, treatment with DNase I) for removing contaminating DNA such as genomic DNA and plasmid DNA. It was confirmed by agarose gel electrophoresis (1.5%, Tris-borate-EDTA buffer) that the sample contained a trace amount of contaminating DNA. The total RNA concentration was measured by spectroscopic analysis.

5. RNA-primed RCA detection
Total RNA isolated from Escherichia coli BL21 (DE3) strain carrying GFP mRNA or pET-AcGFP transcribed in vitro was converted into 10 pmol of circularized oligonucleotides (P1 (SEQ ID NO: 1) and P2 (SEQ ID NO: 2) in Table 1). , together with P3 (SEQ ID NO: 3) or P4 (SEQ ID NO: 4)), 50mM Tris-HCl (pH7.5), 10mM MgCl 2, 20mM (NH 4) were mixed in buffer containing 2 SO ₄ and 10 mM KCl, 100 μl of reaction mixture was obtained. Hybridization of mRNA and circularized oligonucleotide was facilitated by incubation at 95 ° C. for 5 minutes, followed by rapid cooling to 30 ° C. The RCA reaction was performed by adding 100 μl of the hybridized mixture to 50 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 20 mM (NH ₄ ) ₂ SO ₄ , 10 mM KCl, 0.4 mM deoxynucleoside triphosphates (dNTPs), It was started by mixing 100 μl of a reaction solution containing 8 mM dithiothreitol (DTT), SYBR GreenII (Invitrogen) and appropriate units of RepliPHI phi29 polymerase (Epicentre). The fluorescence of SYBR Green II when bound to ssDNA (single-stranded DNA) synthesized by phi29 polymerase was detected with a Qubit fluorometer every 15 minutes. After incubation at 30 ° C. for 15 hours, 15 μl of the synthetic DNA was digested with 20 units of S1 nuclease (Takara Bio Inc.) at 37 ° C. for 30 minutes, and agarose gel electrophoresis (1.0%, Tris-Borate-EDTA buffer) Were continuously analyzed and stained with ethidium bromide for visualization.

6). RNA-primed RCA detection using RNase H
GFP mRNA transcribed in vitro was mixed with 10 pmol of circularized oligonucleotide (P5 in Table 1, SEQ ID NO: 5), 50 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 20 mM (NH ₄ ) ₂ SO ₄ and Mixing in a buffer containing 10 mM KCl yielded 100 μL of reaction mixture. Hybridization of mRNA and circularized oligonucleotide was facilitated by incubation at 95 ° C. for 5 minutes and then rapidly cooled to 30 ° C. The RCA reaction was performed using 100 μL of the hybridized mixture, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 20 mM (NH ₄ ) ₂ SO ₄ and 10 mM KCl, 0.4 mM dNTPs, 8 mM dithiothreitol (DTT). ) And 100 μL of the reaction solution containing an appropriate number of units of RNase H and RepliPHI phi29 polymerase (Epicentre) was mixed. After incubation at 30 ° C. for 15 hours, 15 μL of the synthetic DNA was digested with 20 units of S1 nuclease (Takara Bio Inc.) at 37 ° C. for 30 minutes, and agarose gel electrophoresis (1.0%, Tris-Borate-EDTA buffer) Analyzed continuously, stained with ethidium bromide and visualized

7). Detection of contaminating DNA in RNA samples by PCR
Total RNA was extracted from E. coli BL21 (DE3) strain carrying pET-AcGFP. At this time, the expression of GFP was controlled by automatic induction, and two types of cells were used from the cells that had been induced to induce expression and cells that had not been induced to induce expression using Trizol (registered trademark) Max Bacterial RNA Isolation kit (Invitrogen). Each RNA was prepared. In addition, DNase treatment was carried out by the following procedure for a part (10 μg) of the obtained total RNA sample. 10 μg of total RNA sample was placed in 1 × DNase buffer (50 mM Tris-HCl (pH 8.0), 10 mM MgSO ₄ , 1 mM CaCl ₂ ) with 10 units of RNase-free DNase I (Takara Bio) at 37 ° C. After digestion for 30 minutes, the mixture was separated into a DNase fraction and a nucleic acid fraction by phenol chloroform extraction, and RNA was recovered as a precipitate by ethanol precipitation. The recovered RNA was dissolved in sterilized distilled water treated with diethylpyrocarbonate, and the RNA concentration was determined using the Quant-iT RNA BR Assay Kit.

Detection of contaminating DNA (pET-AcGFP) by PCR was performed according to the following procedure. Prepare a reaction solution containing 20 ng of each RNA sample, 1 × AmpliTaq Gold buffer, 0.2 mM dNTPs, 1 μM of each primer combination (Table 2), 2 mM MgCl ₂ , and 0.625 units of AmpliTaq Gold polymerase at 95 ° C. After 10 minutes of preheating at 95 ° C for 15 seconds, 60 ° C for 30 seconds, 72 ° C for 40 seconds, 35 cycles, followed by 7 minutes pre-extension at 72 ° C and then held at 4 ° C did. As a positive control (P ₁ , P ₂ , P ₃ ) in each primer combination, 1 ng of pAcGFP-1 (Clontech) was used as a template instead of the RNA sample. 2 μL of each PCR product was continuously analyzed using agarose gel electrophoresis (2.0%, Tris-Borate-EDTA buffer), and visualized by staining with ethidium bromide.

8). Detection of RNA by RNA-primed RCA in contaminated DNA-containing sample Using the RNA sample in which the presence of contaminated DNA was confirmed by PCR in (7) above, GFP mRNA was detected by RPRCA of the present invention as follows. 20 ng of each RNA sample was mixed with 10 pmol of circularized oligonucleotide (P4 (SEQ ID NO: 4) in Table 1), 50 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 20 mM (NH ₄ ) ₂ SO ₄ and 10 mM KCl. Mix in buffer containing 10 μl of reaction mixture. As a negative control (N), a reaction mixture containing no RNA sample was prepared. Hybridization of mRNA and circularized oligonucleotide was facilitated by incubation at 95 ° C. for 5 minutes, followed by rapid cooling to 30 ° C. In the RCA reaction, 10 μl of the hybridized mixture was mixed with 50 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 20 mM (NH ₄ ) ₂ SO ₄ , 10 mM KCl, 0.4 mM deoxynucleoside triphosphates (dNTPs), 8 mM. It was started by mixing dithiothreitol (DTT) and 10 μl of reaction containing 10 units of RepliPHI phi29 polymerase (Epicentre). The reaction was carried out at 30 ° C. for 4 hours, and then 20 μl of the synthetic DNA was digested with 24 units of S1 nuclease (Takara Bio) at 37 ° C. for 30 minutes, and 10 μl of the digested solution was subjected to agarose gel electrophoresis ( 1.0%, Tris-Borate-EDTA buffer), and stained with ethidium bromide for visualization.

[Summary of experimental results]
1. Strategy for real-time detection of mRNA using RCA with circularization probe FIG. 1 shows the basic scheme of mRNA detection by RCA. Such a scheme includes the following five steps:
(1) cyclizing a ssDNA probe having a sequence complementary to the target mRNA by intramolecular ligation using CricLigase or the like;
(2) a step of removing unligated ssDNA probe by degradation using exonuclease I in order to avoid non-specific by-product DNA synthesis in continuous RCA reaction;
(3) a step of hybridizing a circular ssDNA probe with the 3 ′ end of the target mRNA,
(4) A step of performing RNA-primed RCA (RCA using RNA as a primer; RPRCA) from an RNA-DNA hybrid by phi29 DNA polymerase;
(5) A step of obtaining fluorescence by binding of ssDNA, which is a result synthesized by RCA, with a fluorescent dye for ssDNA such as SYBR GreenII.
Thereby, real-time detection of mRNA via RPRCA is achieved.

2. Real-time detection of mRNA transcribed in vitro First, it was examined whether phi29 DNA polymerase can adapt mRNA having a long sequence as a primer for RCA reaction. Plasmid pET-AcGFP was digested with NotI, the region between the T7 terminator and the stop codon of the GFP gene, and the corresponding mRNA was prepared by in vitro transcription with T7 RNA polymerase (FIG. 2A). A circular ssDNA probe (P1 or P2) was also used in combination with in vitro transcribed mRNA for RPRCA. The DNA sequence of P1 is complementary to the 3 ′ end sequence of GFP (Table 1). On the other hand, the sequence of P2 has the same length and GC content as P1, but differs from the GFP mRNA in the sequence (Table 1).

  The RCA reaction by phi29 DNA polymerase did not start with mRNA without P1 or P2, and incubation of P2 in the reaction buffer did not cause an RCA reaction in the presence or absence of mRNA (FIG. 2B). And C). However, despite the fact that P1 alone does not affect the RCA reaction, the combination of mRNA and P1 promoted the RCA reaction. From this, it was found that RPRCA enables real-time detection of target mRNA using a ssDNA probe specific for the 3 ′ end of in vitro transcribed mRNA, and that nonspecific circular probes do not affect RPRCA (FIG. 2B and C). These suggest that phi29 polymerase can specifically act on RCA reactions using mRNA as a primer in the presence of an appropriate circular ssDNA probe. Furthermore, the circular ssDNA probe treated with exonuclease I did not produce non-specific by-products after a long reaction period in RPRCA. This indicates that exonuclease I treatment in circular probe preparation contributes to specific and reliable amplification by RPRCA.

  The effect of the length of the circular ssDNA probe in RPRCA was performed using the ssDNA probe P3 (Table 1) covering the DNA region complementary to the 3 'end of GFP mRNA. Interestingly, as shown in FIG. 2D, a sufficient RCA reaction was observed with a long probe (P1), not a short probe (P3), despite having a DNA sequence specific for the same length of GFP mRNA. It was done. This indicates that the length of the probe is related to the initial hybridization of the mRNA and the circular probe and affects the efficiency of RPRCA.

  In summary, the above evidence shows that the RPRCA of the present invention allows the real-time detection of the target mRNA without the generation of irregular DNA amplification by-products using the appropriate circular probe. Yes.

3. RPRCA Sensitivity To clarify the correlation between sensitivity and phi29 DNA polymerase concentration, the effect of concentration on the RPRCA reaction was examined. The RCA reaction was observed when 3.2 ng of in vitro transcribed mRNA was added as a primer for each sample under conditions where the concentration of phi29 DNA polymerase was changed. It was confirmed that the RCA reaction was promoted in a concentration-dependent manner (FIG. 3A). The sensitivity of mRNA detection by RPRCA was then measured at the highest enzyme concentration tested in FIG. 3A (100 units per reaction). As a result, RPRCA was able to detect 320 pg of in vitro transcribed mRNA in 2 hours.

4). Specific detection of target mRNA derived from living microorganisms Whether or not mRNA of living microorganisms could be detected for the purpose by RPRCA was examined. Since induced GFP expression can be easily observed (FIG. 4A), E. coli BL21 (DE3) strain transformed with pET-AcGFP was used as a model for RPRCA. In living cells, the primary structure of GFP mRNA transcribed by T7 RNA polymerase is composed of a 7 base 5 ′ untranslated region (UTR), followed by GFP mRNA, followed by 48 base contiguous residues of 3 ′ UTR (FIG. 4B). In this experiment, ssDNA probe P4 was synthesized. The P4 is synthesized so as to include a DNA sequence corresponding to the 3 ′ end shown underlined in FIG. 4B (Table 1). In vegetatively growing E. coli, the majority of total RNA is occupied by rRNA and tRNA, so both RNAs can affect RPRCA. To examine how much rRNA and tRNA affected RPRCA, total RNA was used for RPRCA after extraction from both induced and non-induced E. coli. When 200 ng of total RNA derived from E. coli with or without induction was incubated with P2 or P4, sufficient RPRCA was observed only for the combination of induced total RNA from E. coli and the circular probe P4 (FIG. 4C). RCA reaction did not occur with total RNA alone or circular probe alone. Furthermore, 2 hours of RPRCA using serially diluted total RNA revealed that GFP mRNA was detectable from 10 ng total RNA (FIG. 4D). Considering that the total amount of RNA (400 μg) was extracted from E. coli with a density of 1.5 × 10 ¹⁰ cells, 10 ng of total RNA corresponds to approximately 4 × 10 ⁵ cells. These results indicate that RPRCA using a combination of bacterial total RNA and the appropriate ssDNA probe not only specifically detected GFP mRNA but also expressed, as in the case of in vitro transcribed mRNA (FIG. 2). It has been shown that GFP mRNA can be clearly distinguished from other RNA species such as rRNA and tRNA that constitute the majority of total RNA.

  Moreover, even if contaminating DNA was contained in the sample (even if the DNA in the sample was not decomposed), it was shown that RNA could be detected satisfactorily. That is, even if the DNA in the sample is not decomposed, an irregular DNA amplification product is produced by contaminating DNA in the sample in which the target RNA is not expressed (“−” in FIG. 4 (negative control)). As is apparent from the fact that this was not the case, a high signal-to-noise ratio (S / N ratio) could be realized in this method.

5. Strategy for RNA detection using RPCA RNase H FIG. 5 shows the basic scheme of RNA detection by RCA including the RNase H treatment step. Such a scheme includes the following six steps:
(1) cyclizing a ssDNA probe having a sequence complementary to the target mRNA by intramolecular ligation using CricLigase or the like;
(2) a step of decomposing and removing ligated ssDNA probe with exonuclease I in order to avoid non-specific by-product DNA synthesis in continuous RCA reaction,
(3) a step of hybridizing a target RNA to a circular ssDNA probe,
(4) a step of forming a nick site with RNase H on the target RNA in the region hybridized with the circular ssDNA probe;
(5) performing RPRCA from the 3 ′ end of RNA at the nicking site with phi29 DNA polymerase;
(6) A step of detecting ssDNA which is a result synthesized by RCA.

6). Detection of mRNA transcribed in vitro by RPRCA using RNAase H When a DNA probe complementary to the internal region of GFP mRNA was used, it was examined whether an RPRCA reaction could occur. Plasmid pET-AcGFP was digested with NotI, the region between the T7 terminator and the stop codon of the GFP gene, and the corresponding mRNA was prepared by in vitro transcription with T7 RNA polymerase (FIG. 6). The transcribed GFP mRNA was used in combination with a circular ssDNA probe (P5). The DNA sequence of probe P1 is complementary to the internal region of GFP (FIG. 6).

In the absence of a circularized single-stranded probe, the RCA reaction by phi29 DNA polymerase was not promoted and no DNA product could be confirmed (FIG. 7). When the probe P5 was used, in the RPRCA reaction under the condition where RNase H was not added, unlike the report of Non-Patent Document 8, no DNA product could be confirmed (FIG. 7). This indicates that the RPRCA method including the RNase H treatment step of the present application can detect the target RNA even if the internal sequence of the target RNA is hybridized with a DNA probe. Further, unlike Patent Document 4, the present invention, which is detected as a single band in the gel, confirms that non-specific by-products are not generated, and its high detection accuracy is recognized.
Moreover, as a result of examining the concentration dependence of RNase H to be added, it was shown that the RPRCA reaction was promoted by the addition of RNase H.

  The above evidence shows that the RNA detection method of the present invention is treated with an appropriate concentration of RNase H in the RPRCA step, so that it does not depend on the information on the 3 ′ end sequence of the target RNA and is nonspecific DNA. It shows that specific detection is possible without producing amplification by-products.

7). Detection of contaminating DNA in RNA samples by PCR Using 3 sets of primers specific to the GFP gene (Table 2), contaminating DNA in RNA samples was detected by PCR. At that time, the expression of GFP was controlled by automatic induction, and two types of total RNA (+) prepared from the cells whose expression was induced and total RNA (-) prepared from the cells whose expression was not induced were obtained. Further, a portion of each was subjected to DNA degradation treatment using DNase I, and total RNAs with or without expression induction, with or without DNase treatment, were obtained. Then, detection of contaminating DNA was attempted using each total RNA.

From any RNA sample that has not been treated with DNase, the amplification product of the target DNA, regardless of the induction or non-induction of GFP, in each primer set is the positive control (P ₁ , P ₂ , P ₃ ) of each primer set. The same was confirmed (FIG. 8). On the other hand, from the RNA sample that had been treated with DNase, the target DNA amplification product was clearly reduced both induced and non-induced (FIG. 8). From the above, it was shown that most of the contaminating DNA can be removed by DNase treatment. However, since a slight band was detected even after DNase treatment, it was also shown that it was difficult to completely remove contaminating DNA from an RNA sample by general DNase treatment alone. Those skilled in the art can understand that this contaminated DNA is also likely to be detected as noise in RT-PCR.

8). Detection of RNA by RNA-primed RCA in contaminated DNA-containing sample Using the RNA sample with or without DNase treatment described in (7) above, detection of GFP mRNA by RPRCA was attempted. As a result, regardless of the presence or absence of DNase treatment, that is, the amount of contaminating DNA, a sufficient amount of DNA amplified by RPRCA was confirmed from the RNA sample prepared from the expression-induced cells (FIG. 9). In addition, regardless of the presence or absence of DNase treatment, no DNA amplification by RPRCA was observed in RNA samples prepared from cells that did not induce expression (FIG. 9). As described above, in the RPRCA of the present invention, only the target RNA is detected with a high S / N ratio without removing contaminating DNA from the RNA sample and without being affected by the amount of contaminating DNA in the RNA sample. It was shown that it can be done.

  The present invention applies RNA-primed rolling circle amplification (RPRCA) using strand displacement DNA synthase, and is affected by RNA type, 3 ′ end sequence information, and the like. In addition, it is related to a method that can detect RNA directly in situ or in real time from samples containing contaminants derived from living organisms in a simple operation and without going through reverse transcription. Research, medical / clinical testing It contributes to a wide range of applications such as the food industry.

Claims (18)

A method for detecting RNA in a sample containing contaminants derived from an organism,
(1) circularizing single-stranded DNA having a sequence complementary to RNA to be detected to produce a circularized single-stranded DNA probe;
(2) a step of hybridizing the circularized single-stranded DNA probe obtained in (1) above with RNA in the sample,
(3) A step of synthesizing single-stranded DNA from the RNA-DNA hybrid obtained in the above (2) by rolling circle amplification (RCA) using RNA as a primer,
(4) The method comprising the steps of adding a label that binds to the single-stranded DNA synthesized in (3) and detecting the RNA to be detected by detecting the label.   The method according to claim 1, wherein the biological contaminant contains DNA.   The method according to claim 1 or 2, wherein the sample is a nucleic acid sample.   The method according to claim 2 or 3, wherein the DNA in the sample has not been decomposed.   The method according to any one of claims 1 to 4, comprising decomposing unreacted single-stranded DNA that has not been circularized in (1).   The method according to any one of claims 1 to 5, wherein the circularized single-stranded DNA probe comprises only a sequence complementary to the RNA to be detected.   The method according to any one of claims 1 to 6, wherein the circularized single-stranded DNA probe has a length of 30 to 4500 bases. After (2) above
(5) The method according to any one of claims 1 to 7, further comprising a step of forming a nick only in the RNA strand of the RNA / DNA hybrid by RNase H.   The method according to claim 8, wherein (3) and / or (5) is performed at a constant temperature in the range of 25C to 65C. 10. The method according to claim 8 or 9, wherein in (5), RNase H is used at a final concentration of 1 × 10 ⁻⁴ to 10 units / reaction. The method according to claim 8 or 9, wherein in (5), RNase H is used at a final concentration of 1 × 10 ^{−7 to} 0.1 unit / μl.   The method according to any one of claims 1 to 11, wherein the RNA is derived from a eukaryote, a prokaryote, a virus or a viroid.   13. The method of claim 12, wherein the RNA has a poly A sequence.   The method of claim 12, wherein the RNA is non-coding RNA.   A method for detecting eukaryotes, prokaryotes, viruses or viroids, comprising the method for detecting RNA according to any one of claims 1 to 14.   The method according to claim 15, wherein microorganisms are detected.   The method for detecting RNA according to any one of claims 1 to 14, and / or the method for detecting eukaryote, prokaryote, virus or viroid according to claim 15, and / or the method according to claim 16. A kit used for a microorganism detection method, comprising a circularized single-stranded DNA probe containing a sequence complementary to RNA to be detected.   The kit according to claim 17, further comprising one or more selected from the group consisting of a deoxynucleotide consisting of adenine, guanine, cytosine and thymine, a reaction buffer and a strand displacement DNA synthase.