WO2006088165A1 - Gene detection reagent and the use thereof - Google Patents

Abstract

It is intended to provide a gene detection reagent which comprises a region that can be hybridized to a target nucleotide sequence, a ribosome binding region, a start codon, and a region encoding a reporter protein characterized in that a conformational change is induced by being hybridized to a target nucleic acid and translation to the reporter protein is initiated. The target nucleic acid in a test sample is detected by mixing the test sample and the gene detection reagent and comparing the expression level of the reporter protein with the case where the test sample is not present.

Description

 Specification

 Gene detection reagent and use thereof

 Technical field

 [0001] The present invention relates to a gene detection reagent, kit, and gene detection method for use in examining the presence of a foreign gene or structural abnormality of a gene.

 Background art

 [0002] Genetic testing can be divided into those that examine the presence of foreign genes that do not exist in the self and those that analyze genetic structural abnormalities. Examples of testing for the presence of foreign genes include identification of microorganisms that are difficult to culture and detection of pathogenic microorganisms during antibiotic treatment and early infection. Examples of genetic structural abnormality tests include tests for definitive diagnosis of genetic diseases, prediction tests for genetic diseases, tests that do not affect phenotype, and DNA polymorphism tests (parent-child test and forensic test). .

 [0003] Conventionally, PCR was performed on a test sample using a PCR primer capable of amplifying the target gene, and the presence or mutation of the gene was detected by a method of sequencing the PCR product.

 [0004] In recent years, a reagent capable of genetic diagnosis in the state of the cell without extracting the gene from the cell has been proposed, and a typical example thereof is a molecular beacon. As such a reagent, for example, an oligonucleotide comprising a loop portion complementary to a target sequence and stem-forming sequences present at both ends thereof, a fluorescent dye is bound to one end, and a quencher is bound to the other end. (Non-patent document 1).

When this reagent is not hybridized with the target nucleic acid and irradiated with light, the fluorescent dye at both ends and the quencher are close to each other, so that the fluorescence emitted from the fluorescent dye transfers energy to the quencher to produce fluorescence. It does not emit light or emits light having a wavelength different from the original fluorescence. On the other hand, when the target nucleic acid is hybridized to the loop part, the loop part becomes double-stranded and rigid, so the stem forming part dissociates. As a result, the fluorescent dye and the quencher are separated, and the fluorescent substance inherent in the fluorescent material is emitted. Therefore, by detecting fluorescence intensity or wavelength. Thus, it is possible to detect whether or not the target sequence exists. Figure 8 shows how the target sequence is detected by the molecular beacon of this document.

 [0005] However, since this method detects the light emitted by the fluorescent dye bound to the molecular beacon as it is, the detection sensitivity is low unless a large amount of molecular beacons are used.

 [0006] In order to detect whether or not the target nucleic acid has hybridized to the loop, a molecular bicon using a DNAzyme Z ribozyme has been proposed in addition to the combination of a fluorescent dye and a quencher ( Non-patent document 2). Figure 9 shows a method for detecting a target nucleic acid using this molecular beacon. The DNAzyme has two regions complementary to the target nucleic acid sequence on both sides of the loop portion that exhibits catalytic activity bound to a divalent metal ion, and hybridizes with the target nucleic acid in these two regions. It is thought to cut it at the loop. The molecular beacon described in this document has a stem-loop structure formed using such a target binding region of one of the DNAzymes. The loop portion has a sequence complementary to the target nucleic acid sequence.

 [0007] Since this molecule forms a stem loop structure in the absence of the target nucleic acid, the DNAzyme cannot bind to the target nucleic acid, and the cleavage activity is not expressed. On the other hand, when the target nucleic acid is hybridized in the loop region, the loop opens, and the DNA zym binds to the target nucleic acid and the cleavage activity is expressed. By detecting this cleavage activity, the presence or absence of a sequence that can hybridize to the loop portion can be confirmed.

 However, this method also has low detection sensitivity because it detects the cleavage activity of the molecular beacon molecule itself.

[0009] As a molecular beacon with high detection sensitivity, Non-Patent Document 3 reports that an enzyme is bound to an inhibitor via a single-stranded DNA fragment. This molecular beacon contains a sequence complementary to the target nucleic acid sequence in the single-stranded DNA fragment, and when the target nucleic acid is not present, the single-stranded DNA fragment is flexible, so that the inhibitor interacts with the enzyme. Although it is bound to the site, when the target nucleic acid and the DNA fragment are hybridized, the part becomes rigid and extends, so that the inhibitor is removed from the enzyme and the enzyme activity is expressed. Since this molecular beacon detects the conversion of the substrate by the enzyme, the detection sensitivity is increased accordingly. [0010] However, this molecule requires time and labor to bind dissimilar substances such as enzyme proteins, nucleic acids, and low molecular weight compounds.

 [0011] In addition, Patent Document 1 uses RNA called a riboregulator to control the on / off of translation start of a specific gene to control the expression of the specific protein and elucidate the function of the protein in vivo. Tell me how to do it. The riboregulator in this document is an mRNA molecule containing a ribosome binding region (RBS), an initiation codon, and a green fluorescent protein coding region, and having a “cis repressive RNA” region upstream of RBS.

 The cis-repressive RNA region consists of a sequence that can hybridize with RBS within the molecule. Normally, since this region and RBS are hybridized, the ribosome does not bind to RBS and is not translated into green fluorescent protein. Furthermore, cis-reactive RNA and RBS are dissociated by the “transactivating RNA” having a complementary region in the cis-reactive RNA region and hybridized to the cis-reactive RNA, which translates into a green fluorescent protein. Is called.

 [0012] According to the method of this document, the transactivating RNA must have the same sequence as the ribosome binding region. Therefore, this riboregulator cannot detect the presence of an arbitrary base sequence.

 Non-patent literature l: Tyagi, S .; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303.

 Non-Patent Document 2: Stojanovic MN. Et al. ChemBioChem 2001, 2, 411

 Non-Patent Document 3: Saghatelian, A. et al. J. Am. Chem. So 2003, 125, 344.

 Patent Document 1: WO2004 / 046321

 Disclosure of the invention

 Problems to be solved by the invention

[0013] An object of the present invention is to provide a highly sensitive gene detection reagent comprising a nucleic acid, a kit including the reagent, and a method capable of detecting the presence or absence of a target gene with high sensitivity using a nucleic acid. .

 Means for solving the problem

[0014] In order to solve the above problems, the present inventors have conducted research and obtained the following knowledge.

(0 Region that can hybridize with target nucleic acid sequence, ribosome binding region, start codon, and RNA that has a region that encodes a reporter protein, and whose conformational change induced by hybridization between the target nucleic acid and its complementary region initiates translation into the reporter protein, detects the expression of the reporter protein Therefore, the target nucleic acid can be detected and used as a gene detection reagent.

 (ii) For example, a ribosome binding region (hereinafter also referred to as “RBS”), a start codon, and a region encoding a reporter protein are provided in order from upstream, and the target nucleic acid sequence is hybridized upstream of the ribosome binding region. In the absence of a target nucleic acid, an RNA comprising a region (B) that can hybridize with a region containing a ribosome binding region via a region (A) can be hybridized with the region (B) and RBS. ) Is a loop- stem structure. Further, in the presence of the target nucleic acid, the target nucleic acid and the region (A) are hybridized, so that this region becomes rigid and the loop extends, and as a result, the RBS and the region (B) are dissociated. This allows RBS to bind to RBS and initiates translation of the reporter gene.

 [0015] By utilizing this phenomenon and detecting the presence or absence of expression of the reporter gene, the presence or absence of the target nucleic acid or the single nucleotide polymorphism in the test sample can be determined.

 [0016] The present invention has been completed based on the above findings, and provides the following gene detection reagents, gene detection kits, and gene detection methods.

 [0017] Item 1. A gene detection reagent comprising a region capable of hybridizing with a target nucleic acid sequence, a ribosome binding region, an initiation codon, and a region encoding a reporter protein, wherein higher-order structural changes are caused by hybridization with the target nucleic acid. A reagent for gene detection that is induced and begins to translate into a reporter protein.

 [0018] Item 2. The gene detection reagent according to Item 1, which has at least a region strength NA that codes at least a ribosome binding region, an initiation codon, and a reporter protein.

 [0019] Item 3. The ribosome cannot bind to the ribosome binding region, and the conformation change induced by hybridization between the target nucleic acid and the region that can hybridize with the target nucleic acid. Item 3. The gene detection reagent according to Item 2, which enables translation into a reporter protein.

[0020] Item 4. In order from upstream, ribosome binding region, start codon, and reporter protein The gene detection according to Item 3, further comprising a region capable of hybridizing with a region including the ribosome binding region via a region capable of hybridizing with the target nucleic acid sequence further upstream of the ribosome binding region. Reagent.

[0021] Item 5. The gene detection according to Item 4, wherein the ratio of the number of bases in the region hybridizable with the target nucleic acid sequence to the number of bases in the region hybridizable with the region including the ribosome binding region is 1 to 10 reagent.

[0022] Item 6. The gene detection reagent according to Item 4, wherein a region capable of hybridizing with a region containing a ribosome binding region comprises 5 to 30 bases, and a region capable of hybridizing with a target nucleic acid sequence comprises 5 to 60 bases.

 [0023] Item 7. The gene detection reagent according to Item 1, wherein the region capable of hybridizing with the target nucleic acid sequence is a region complementary to the target nucleic acid sequence.

[0024] Item 8. The gene detection reagent according to Item 1, comprising a region that functions as a ribozyme that cleaves when hybridized with a target nucleic acid.

[0025] Item 9. The gene detection reagent according to Item 1, wherein the reporter protein is an enzyme.

Item 10. The reagent for gene detection according to Item 9, wherein the enzyme is involved in the production of a substance that generates visible light.

[0027] Item 11. A region capable of hybridizing with a target nucleic acid sequence, a ribosome binding region, an initiation codon, and a region encoding a reporter protein, wherein a conformational change induced by hybridization with the target nucleic acid is induced in the reporter protein. A gene detection reagent containing DNA that can be transcribed into RNA that initiates translation.

Item 12. The RNA force S is in a state where ribosome cannot bind to the ribosome binding region.

Section 11, which enables the ribosome to bind to the higher-order structurally modified ribosome binding region induced by hybridization between the target nucleic acid and the target nucleic acid hybridizing region, thereby initiating translation into the reporter protein. The reagent for gene detection described in 1.

[0029] Item 13. The RNA comprises a region encoding a ribosome binding region, an initiation codon, and a reporter protein in order from upstream, and a region capable of hybridizing with a target nucleic acid sequence further upstream of the ribosome binding region. Item 13. The reagent for gene detection according to Item 12, comprising a region containing a ribosome binding region and a region that can be hybridized. [0030] Item 14. The ratio of the number of bases in a region capable of hybridizing to a target nucleic acid sequence to the number of bases in a region capable of hybridizing with a region including a ribosome binding region is 1 to 10.

4. The gene detection reagent according to 3.

Item 15. The reagent for gene detection according to Item 13, wherein the region capable of hybridizing with the region including the ribosome binding region has a strength of 5 to 30 bases, and the region capable of hybridizing with the target nucleic acid sequence has 5 to 60 bases.

[0032] Item 16. The gene detection reagent according to Item 11, wherein the region capable of hybridizing with the target nucleic acid sequence is a region complementary to the target nucleic acid sequence.

[0033] Item 17. The reagent for gene detection according to Item 11, wherein the RNA includes a region that functions as a ribozyme that cleaves when hybridized with a target nucleic acid.

[0034] Item 18. The gene detection reagent according to Item 11, wherein the reporter protein is an enzyme.

Item 19. The gene detection reagent according to Item 18, wherein the enzyme is involved in the production of a substance that generates visible light.

[0036] Item 20. A gene detection kit comprising the gene detection reagent according to Item 1.

[0037] Item 21. The gene detection kit according to Item 20, further comprising a prokaryotic cell extract.

[0038] Item 22. The gene detection kit according to Item 20, further comprising an RNA degrading enzyme that specifically cleaves RNA hybridized with DNA.

[0039] Item 23. A gene detection kit comprising the gene detection reagent according to Item 11.

[0040] Item 24. The gene detection kit according to Item 23, further comprising a prokaryotic cell extract.

[0041] Item 25. The gene detection kit according to Item 23, further comprising an RNA degrading enzyme that specifically cleaves RNA hybridized with DNA.

[0042] Item 26. First step of mixing the test sample with the gene detection reagent according to Item 1, and the expression level of the reporter protein from the gene detection reagent in the absence of the test sample.

A gene detection method comprising a second step of determining the presence or absence of a target nucleic acid in a test sample by comparing the expression level of a reporter protein from the gene detection reagent according to 1.

[0043] Item 27. The method according to Item 26, wherein the reporter protein is expressed from the gene detection reagent in the presence of a prokaryotic cell extract.

[0044] Item 28. In the first step, the test sample, the gene detection reagent according to Item 1, and DN Item 27. The method according to Item 26, comprising the step of mixing A with an RNase that specifically cleaves the hybridized RNA.

[0045] Item 29. The first step of mixing the test sample with the gene detection reagent according to Item 11, and the expression level of the reporter protein from the gene detection reagent in the absence of the test sample. A gene detection method comprising a second step of determining the presence or absence of a target nucleic acid in a test sample by comparing with the expression level of a reporter protein from the gene detection reagent according to Item 11.

 [0046] Item 30. The method according to Item 29, wherein the reporter protein is expressed from the gene detection reagent in the presence of a prokaryotic cell extract.

[0047] Item 31. In the first step, the test sample, the gene detection reagent according to Item 1, and DN

Item 30. The method according to Item 29, comprising a step of mixing A with an RNase that specifically cleaves the hybridized RNA.

[0048] Item 32. A first step of mixing a test sample, the gene detection reagent according to Item 1, and an RNase that specifically cleaves RNA hybridized with DNA, and a gene detection reagent The presence or absence of the target nucleic acid in the test sample is determined by comparing the expression level of the reporter protein with the expression level of the reporter protein from the gene detection reagent according to Item 1 in the absence of the test sample. A method for detecting a single nucleotide polymorphism comprising the second step.

[0049] Item 33. A first step of mixing a test sample, the gene detection reagent according to Item 11, and an RNase that specifically cleaves RNA hybridized with DNA, and a gene detection reagent The presence or absence of the target nucleic acid in the test sample is determined by comparing the expression level of the reporter protein with the expression level of the reporter protein from the gene detection reagent described in Item 11 in the absence of the test sample. A method for detecting a single nucleotide polymorphism comprising the second step.

 [0050] Item 34. A nucleic acid comprising a region capable of hybridizing with a target nucleic acid sequence, a ribosome-binding region, an initiation codon, and a region encoding a reporter protein, wherein a conformation change is induced by hybridization with the target nucleic acid. And use as a reagent for gene detection of a nucleic acid that begins translation into a reporter protein.

[0051] Item 35. RNA comprising a region capable of hybridizing with a target nucleic acid sequence, a ribosome binding region, an initiation codon, and a region encoding a reporter protein, Use as a reagent for gene detection of DNA that can be transcribed into RNA that initiates translation into a reporter protein by a conformational change induced by hybridisation.

 The invention's effect

 [0052] According to the gene detection reagent comprising RNA, which is the first reagent of the present invention, the presence or absence of the target nucleic acid in the test sample is the presence or absence of translation from the RNA encoding the reporter protein into the reporter protein. appear. This translation makes it possible to produce, for example, a multi-molecule reporter protein from one molecule of RNA, so that detection signals such as fluorescence can be amplified and detection sensitivity can be improved. Therefore, when the reagent of the present invention is used, it is not always necessary to amplify the nucleic acid in the test sample in advance by PCR or the like, and a cell lysate or the like can be directly used as the test sample.

 [0053] In addition, according to the gene detection reagent comprising DNA, which is the second reagent of the present invention, a transcription process to DNA force RNA is added, and multiple molecules of RNA are generated from one DNA molecule. Also, since DNA is very stable, it is easy to use as a reagent.

 [0054] Further, when an enzyme such as luciferase or β-galactosidase is used as the reporter protein, the detection sensitivity is further enhanced because a plurality of substrates are converted from one molecule of enzyme.

 [0055] In addition, in the case of using an RNase that specifically cleaves RNA in a DNA-RNΑ double strand hybridized with DNA together with the gene detection reagent of the present invention, the first of the present invention When the RNA of the reagent or the transcription product RNA of the second reagent RNA and the target nucleic acid (usually DNA) are hybridized, the RNA strand on the reagent side is cleaved and separated from the target nucleic acid. The target nucleic acid can further bind to another RNA molecule, and similarly forms a cycle of cleavage of the reagent RNA and detachment from the target nucleic acid by the action of an enzyme. In this way, the expression of one reporter gene can be amplified by successively hybridizing one molecule of the target nucleic acid with the reagent of the present invention. For this reason, detection sensitivity is further improved, and even when the target nucleic acid exists only in femtomolar order, gene detection can be performed without amplifying the target nucleic acid by PCR or the like.

[0056] Here, the base sequence selectivity decreases as the region that can be hybridized with the target nucleic acid sequence becomes longer, that is, it can be detected when there is a mismatch. The number of mismatched bases decreases. On the other hand, when shortening the region that can hybridize with the target nucleic acid sequence in the reagent RNA molecule, the force that causes higher-order structural changes is weakened. There is a need. In this case, higher-order structural changes of non-specific RNA molecules are likely to occur. Therefore, when shortening the region that can hybridize with the target nucleic acid sequence, it is necessary to shorten the length of the region that includes the ribosome binding region and the region that can be hybridized, for example, in order to facilitate higher-order structural changes due to hybridization. There is. In this case, the expression of the reporter gene is likely to occur due to nonspecific binding to the ribosomal force S ribosome binding region.

[0057] Under such circumstances, when an RNase that specifically cleaves DNA-RNA double-stranded RNA is used together with the reagent of the present invention, RNA that is bound when the target nucleic acid binds to the reagent RNA molecule. Degradative enzymes cleave this RNA molecule to force conformational changes. That is, the state in which the ribosome binding region and the start codon hybridize with a complementary sequence is eliminated. For this reason, even if the length of the region capable of hybridizing with the target nucleic acid sequence of the reagent RNA molecule is short and the molecular structure hardly undergoes a higher order structural change (for example, even if the region capable of hybridizing with the region including the liposome binding region is lengthened). ), Hybridization will ensure higher-order structural changes. As a result, even a single base mismatch between the target nucleic acid and the region capable of hybridizing with the target nucleic acid can be detected more reliably, that is, a single nucleotide polymorphism can be detected. The selectivity of single-base mismatch is also enhanced by the nature of RNase, which cleaves only perfectly matched base pairs.

 [0058] Also, when the reagent RNA itself is a ribozyme that has the ability to self-cleavage when hybridized with the target nucleic acid, the same effect as that obtained when the RNase is used can be obtained.

 [0059] Such gene detection using the reagent of the present invention can be referred to as "catalytic gene detection".

[0060] Further, the gene detection reagent of the present invention does not contain a synthetic compound such as a fluorescent substance or an enzyme inhibitor, and is composed only of nucleic acid, so that it is easy to manufacture. Moreover, it is only necessary to use the cell extract for use. It is also manufactured and used using only biological materials. Therefore, it can be applied to future living organisms.

Brief Description of Drawings

FIG. 1 is a diagram showing the structure of MB-luciferase mRNA constructed in Example 1.

 [Fig. 2] Chemiluminescence intensity in the presence of flumati oligonucleotides and mismatched oligonucleotides when MB-luciferase mRNA is in the saddle type, in the absence of these targets It is a duff expressed as a relative value to the chemiluminescence intensity.

 [Fig. 3] MB luciferase expression Chemiluminescence intensity in the presence of flumati oligonucleotide and mismatched oligodeoxynucleotide when dsDNA is in a saddle type, chemistry in the absence of these targets It is a graph expressed as a relative value with respect to emission intensity.

 [FIG. 4] Chemiluminescence images of each reaction mixture were measured with Versadoc3000 in the presence of flumati oligonucleotides and mismatched oligonucleotides when MB luciferase-expressed dsDNA was in a saddle type. Is.

[Fig. 5] Fluorescence image of MB-type MB-luciferase DNA transcribed using a cell-free transcription / translation system that does not contain ribosomes and tRNA in the presence of full-matched oligonucleotides and in the absence of target Was measured with an ATTO densitograph.

 FIG. 6 is a graph showing the relationship of chemiluminescence intensity to target amount.

 [Fig. 7] Chemoluminescence intensity in the presence of flumati oligonucleotide and 1-base GT mismatched oligonucleotide when MB-luciferase mRNA is in the saddle type, without target It is the graph represented with the relative value with respect to the chemiluminescence intensity of. Moreover, it is a graph which shows the effect by use of RNaseH.

 [Fig. 8] The chemiluminescence intensity in the presence of flumatsu oligodeoxynucleotide when MB-luciferase mRNA is of the vertical type, relative to the chemiluminescence intensity in the absence of the target. It is the represented graph. Also, it is a graph showing the effect of using RNaseH.

[Fig. 9] Fig. (A) shows the full-maturity of MB-galactosidase dsRNA in the vertical type. It is the graph which represented the light absorbency by nitrophenol in presence of an oligodeoxynucleotide with the relative value with respect to the light absorbency in case a target does not exist. Moreover, it is a graph which shows the effect by use of RNaseH. Fig. (B) shows the result of visual observation of coloration by nitrophenol when β-galactosidase dsDNA is in the form of a cage.

 FIG. 10 is a diagram for explaining gene detection using an example of a conventional molecular beacon.

 FIG. 11 is a diagram for explaining gene detection using another example of a conventional molecular beacon.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0062] Hereinafter, the present invention will be described in detail.

 m¾ 云 chine gold expression

 Reagent consisting of RNA

 The first gene detection reagent of the present invention comprises a region capable of hybridizing with a target nucleic acid sequence, a ribosome binding region, an initiation codon, and a region encoding a reporter protein, and a conformational change induced by hybridization with the target nucleic acid. Is a reagent that initiates translation into a reporter protein.

 [0063] This reagent should have at least a ribosome-binding region, an initiation codon, and a region encoding a reporter protein having RNA power. The other part may be any nucleic acid sequence such as RNA, DNA, or artificial nucleic acid. Examples of the artificial nucleic acid include peptide nucleic acid (PNA; P-marked tide Nucleic Acid), LNA (Locked Nucleic Acid) and the like.

 [0064] The structure of the reagent is not particularly limited as long as the target nucleic acid binds to a region capable of hybridizing with the target nucleic acid, so that a factor necessary for translation initiation can be bound to the reagent. Examples of such a structure include a hairpin structure in which a region capable of hybridizing to a target nucleic acid is a loop and two regions existing on both sides form a stem by base pair formation. Examples of the two regions forming the stem include a region capable of hybridizing with a ribosome binding region and a region including the same, a region capable of hybridizing with an initiation codon and a region including the region, and the like.

[0065] By having such a hairpin structure, the ribosome binding region, the initiation codon, etc. are usually in a double-stranded state, so that factors necessary for translation cannot be bound, and the reporter There is virtually no translation into a single protein. Furthermore, when the target nucleic acid binds to a region that can hybridize with the target nucleic acid, this loop becomes rigid and extends, and as a result, all or part of the double strand of the stem is eliminated, and the factor force S required for translation initiation S It becomes able to bind to the ribosome binding region and initiation codon, and translation into the reporter protein begins.

[0066] As a reagent in which a ribosome-binding region and a region capable of hybridizing with the ribosome-binding region form a double-stranded system, encode a ribosome-binding region, an initiation codon, and a reporter protein in order from the upstream. A reagent comprising a region, and a region further hybridized with a region containing a ribosome binding region via a region capable of hybridizing with a target nucleic acid sequence is provided further upstream of the ribosome binding region. This reagent usually has a target nucleic acid-complementary region that hybridizes with a region that can hybridize with a region that includes the ribosome-binding region in the reagent molecule and a region that can hybridize with this region. It has a hairpin structure with a loop. If the target nucleic acid is present here, it hybridizes with the target nucleic acid complementary region, and the loop extends, resulting in a ribosome binding region force S1 strand state. This conformational change allows the ribosome to bind to the ribosome binding region and initiates translation into the reporter protein.

 [0067] Ribosome binding regions include those that function in prokaryotic cells, those that function in eukaryotic cells, those derived from viruses, and the like. The ribosome-binding region that functions in prokaryotic cells refers to a region consisting of a purine-rich sequence to which ribosomes can bind in prokaryotic cell mRNA, and generally consists of about 3 to 9 bases. The ribosome binding region that functions in prokaryotic cells is also referred to as SD-coordinated IJ. Examples of ribosome binding regions that function in eukaryotic cells include the Kozak sequence (5'—AGCCACCAUGG—3 ′) (SEQ ID NO: 1) and the ribosome internal recognition site (I RES: Internal Ribosome Entry Site). . Examples of the virus-derived ribosome binding region include a ribosome internal recognition site (IRES). The viral I RES is a sequence contained in the viral RNA, which changes the three-dimensional structure of the liposome by binding to the host ribosome and usually initiates translation of the viral protein. With the gene detection reagent of the present invention, translation of a reporter protein can be initiated using viral IRES.

[0068] The number of bases present between the ribosome binding region and the start codon is not limited, but is preferred. 3 ~: It should be about 12 bases away. Within this range, translation initiation starting with ribosome binding is efficiently performed.

 [0069] The initiation codon is usually AUG, but other combinations 1J such as GUG can be used as long as it functions as an initiation codon.

 [0070] The type of reporter protein is not particularly limited, and any known reporter protein can be used without limitation. Known reporter genes include, for example, a luciferase gene, a secreted alkaline phosphatase (SEAP) gene, a chloramphenicol acetyl transferase (CAT) gene, and a j3_galactosidase gene. Since these reporter genes encode enzyme proteins and detect their expression using an enzyme reaction, the detection sensitivity increases accordingly. The reporter gene may be one that detects the signal of the product itself, such as a green fluorescent protein gene. In addition, secretory alkaline phosphatase, chloramphenicol acetyltransferase, and i3-galactosidase are preferable because their substrate products often emit visible light, so that enzyme activity can be easily detected.

 [0071] The region that can hybridize with the region containing RBS may be a region that can hybridize with the RBS in this molecule or a region composed of RBS and the adjacent region. Here, “can hybridize” means to be able to hybridize under the conditions of use of this reagent. For example, after incubation at 37 ° C. in a prokaryotic cell extract and washing at 37 ° C. in the same solution. In addition, a positive hypritizing signal is observed.

 [0072] In particular, it is preferable that this region and RBS are complementary, but mismatches may exist as long as hybridization is possible at the temperature at which the target nucleic acid detection method is performed. For example, if the temperature of this method is 37 ° C and there are 10 or more bases in the iridescence region. There can be a mismatch of more than / ο.

[0073] The length of the region capable of hybridizing with RBS is preferably about 5 to 30 bases, more preferably about 8 to about 10 bases. Even if the target nucleic acid does not exist, this region and RBS are hybridized or single-stranded and are in equilibrium. If the length of this region is in the above range, such nonspecific double-strand dissociation that does not depend on the target nucleic acid hardly occurs. The above Thus, the region that can hybridize with the target nucleic acid sequence has a loop structure in the absence of the target nucleic acid. The loop is extended by the formation of a double strand between this region and the target nucleic acid, thereby forming a stem. All or part of the double-stranded state of the region containing RBS and the region that can hybridize to it is eliminated. Therefore, if the length of the region that can hybridize with the region containing RBS is within the above range, the length of the region that can hybridize with the target nucleic acid sequence does not become too long, and mismatch between them can be detected with high sensitivity.

 [0074] Examples of the target nucleic acid sequence include sequences in genes involved in human diseases when detection for genetic diagnosis of diseases is performed. In the case of detection for parent-child or forensic evaluation, sequences near genetic markers can be mentioned. In addition, when identifying pathogenic microorganisms that have infected humans or detecting foods to detect microbial contamination, chromosome sequences unique to those microorganisms can be mentioned.

 [0075] The region that can hybridize with the target nucleic acid sequence should be hybridized to all or part of the target nucleic acid sequence. Above all, it is preferable that the region is complementary to the target nucleic acid sequence, so that even a single base mismatch can be detected. It may be complementary. The length is preferably about 5 to 60 bases, more preferably about 14 to 20 bases. As described above, this region takes a loop structure in the absence of the target nucleic acid. However, if the length of the region that can hybridize with the target nucleic acid sequence is within the above range, double-stranded formation with the target nucleic acid occurs. By extending the loop, the double-stranded state of RBS can be easily eliminated, and the detection sensitivity of mismatches with the target nucleic acid is increased.

 In any case, the ratio of the number of bases in the region capable of hybridizing with the target nucleic acid to the number of bases in the region containing RBS is preferably about 1 to 10 and more preferably about 2 to 5. When the ratio is within the above range, the stem can be easily opened by binding to the loop portion of the target nucleic acid, and the mismatch detection sensitivity is high when the length of the region that can hybridize with the target nucleic acid sequence is appropriate. Become.

[0077] The region capable of hybridizing with the target nucleic acid sequence may be adjacent to the RBS, and an arbitrary sequence of about! To 5 bases may exist between them. Similarly, the region that can be hybridized with the target nucleic acid sequence may be adjacent to the region that can hybridize with the region containing RBS, or an arbitrary sequence of about 1 to 5 bases may exist between these regions. [0078] In addition, as a reagent in which a start codon and a region capable of hybridizing with the start codon form a double-stranded stem, the ribosome binding region, the start codon, and the reporter protein are encoded in this order from the upstream. And a reagent comprising a region that can be hybridized with a region containing a start codon via a region that can hybridize with a target nucleic acid sequence further upstream of the ribosome binding region.

[0079] In this reagent, the presence of the target nucleic acid makes the initiation codon strength in a double-stranded state, allowing binding of factors necessary for translation initiation, and translation is initiated. The region that can hybridize with the target nucleic acid and the region that can hybridize with the start codon may be adjacent to each other, or an arbitrary sequence of about! To 5 bases may exist between them. Other configurations are the same as those of the reagent having a stem including a ribosome binding region.

[0080] The upper limit of the size of the first reagent molecule of the present invention is not particularly limited as long as it can be translated into a reporter protein using a cell extract, but is usually 1000 ~

If it is about 2000 bases,

[0081] This reagent may contain a region that functions as a ribozyme that cleaves its own RNA strand when hybridized with a target nucleic acid. The self-cleaving position may be a position that initiates translation of the reporter gene by cleaving RNA, but typically includes a position within a region capable of hybridizing with the target nucleic acid sequence. In this case, the region that can hybridize with the target nucleic acid sequence is RNA, and this region functions as a ribozyme. Such RNA includes, for example, a self-cleaving hammer-head type ribozyme having a region capable of hybridizing with a target nucleic acid sequence as a site for binding of a nucleic acid sequence to a hairpin-type ribozyme.

 Reagent consisting of DNA

 The second gene detection reagent of the present invention is a reagent containing DNA that can be transcribed into the first reagent described above and composed of RNA. This DNA is usually double-stranded DNA. This DNA should have a promoter region necessary for transcription upstream of the region that can hybridize with the region containing RBS.

[0082] The force S for which a plasmid containing a reporter gene is commercially available, usually includes a promoter region, a ribosome binding region, an initiation codon, and a reporter gene. Therefore, a DNA sequence that can be transcribed into a region that can hybridize with the region containing RBS or the start codon and a region that can hybridize with the target nucleic acid sequence should be inserted between the promoter region and the ribosome binding region of this commercially available reporter plasmid. Les. This sequence can be chemically synthesized.

[0083] Since the second reagent of the present invention contains DNA that is difficult to be decomposed, it is a reagent with good storage stability.

 αι Kiyun Kit

 The gene detection kit of the present invention is a kit provided with the gene detection reagent of the present invention described above.

 [0084] When the ribosome-binding region functions in prokaryotic cells, a prokaryotic cell extract may be provided. Usually, since a factor necessary for transcription and translation is contained in a test sample, translation into a reporter protein, or transcription and translation is performed by this cell extract. If the ribosome binding region functions in eukaryotic cells or is derived from a virus, use a eukaryotic cell extract.

 [0085] The cell extract may be prepared by the Zubay method (Zubay, G. Ann. Rev. Genet. 1978, 7, 267).

[0086] The kit of the present invention may further comprise an RNase having an activity of specifically cleaving RNA hybridized with DNA. This RNase is preferably one that cleaves RNA in a region where DNA and RNA are completely complementary. As a result, when the region that can hybridize with the target nucleic acid sequence of the reagent RNA is completely complementary to the corresponding region of the DNA that is the target nucleic acid, the RNA in the double strand is cleaved. RNaseH has a very high mismatch recognition ability, so it can reliably detect single nucleotide polymorphisms. This RNase is preferably an endonuclease, whereby the reagent RNA hybridized with the target nucleic acid can be efficiently cleaved and the target nucleic acid can be separated from the reagent RNA. An example of such an RNase is RNaseH. In the gene detection method of the present invention, a test sample and the gene detection reagent of the present invention are mixed. In the test sample by comparing the expression level of the reporter protein from the gene detection reagent with the expression level of the reporter protein from the gene detection reagent of the present invention in the absence of the test sample. And a second step of determining the presence or absence of the target nucleic acid.

 [0087] When the ribosome binding region of the gene detection reagent functions in prokaryotic cells, the reporter protein may be expressed in the presence of a prokaryotic cell extract.

 [0088] Examples of test samples include, but are not limited to, collected cells such as mucosal cells in the oral cavity, blood, saliva, hair, and the like. Foods that are suspected of bacterial infection can also be used.

 [0089] In the first step, the test sample may be used as it is when the nucleic acid is exposed in the sample, but it is usually sufficient to use the cells by crushing or lysing the cells according to a conventional method. In addition, the sensitivity is further improved if the target nucleic acid sequence is amplified by PCR with respect to the cell lysate or cell lysate.

 [0090] The amount of the reagent of the present invention to be used should be about 1 to 10 times in molar ratio with respect to the test sample when the reagent contains RNA. It should be about 0.1 times. In addition, the amount of cell extract used may be about 2.5 to 10 / iL per test sample.

 [0091] After mixing these, it is usually incubated at about 25-42 ° C for about 10-60 minutes.

 [0092] In the first step, when the test sample and the gene detection reagent of the present invention are mixed, an RNA-degrading enzyme that specifically degrades RNA hybridized with DNA can be mixed. Since this RNase does not degrade single-stranded RNA before hybridization, the mixing order thereof is not particularly limited. For example, a mixture of RNase and a gene detection reagent may be added to a test sample. Good.

[0093] By using such an RNase, even a single-base mismatch can be detected, so the method of the present invention is a single-nucleotide polymorphism detection method. In this case, the region that can hybridize with the target nucleic acid sequence in the reagent for gene detection has a sequence complementary to the wild type sequence of the target nucleic acid sequence or the predicted single nucleotide variant sequence, the displacement force, and the like. Let's do it. [0094] In the second step, reporter protein expression may be detected by a method according to the type. The expression of these proteins can be detected and measured using the following commercially available kits.

 For example, the expression of noluciferase is detected using a luminometer, a liquid scintillation counter, a top counter, or the like by adding luciferin (for example, manufactured by Toyo Ink) as a luminescent substrate to the cell lysate and luminescence due to decomposition of the substrate. Just measure. The expression of alkaline phosphatase can be detected and measured using, for example, L x Mi_Phos530 (manufactured by Wako Pure Chemical Industries, Ltd.). The expression of chloramphenicol acetyltransferase can be detected and measured using FAST CAT Chrolam phenicol Acetyltransferase Assay Kit (manufactured by Wako Pure Chemical Industries, Ltd.). β-galactosidase expression can be detected and measured using Aurora Gal-XE (Wako Pure Chemical Industries, Ltd.)

[0095] In the second step, the expression level of the reporter protein may be compared between when the test sample is not present, that is, when the reagent of the present invention is incubated under the same conditions and when the test sample is present. . The amount of protein expression can be compared directly by visual observation. The protein expression level in each case can be measured and compared.

 [0096] The criteria for measuring the protein expression level in each case are not limited thereto. For example, if the protein expression level is improved 3 times or more due to the presence of the test sample, the target nucleic acid sequence in the test sample is determined. Can be determined to exist.

 (IV) Use as a gene extraction reagent

 A first use of the present invention is a nucleic acid comprising a region capable of hybridizing with a target nucleic acid sequence, a ribosome binding region, an initiation codon, and a region encoding a reporter protein, wherein the higher-order structural change is caused by hybridization with the target nucleic acid. Use as a gene detection reagent of a nucleic acid that is induced to initiate translation into a reporter protein.

[0097] A second use of the present invention is RNA comprising a region capable of hybridizing with a target nucleic acid sequence, a ribosome binding region, an initiation codon, and a region encoding a reporter protein, and is highly induced by hybridization with the target nucleic acid. The next structural change is the use of DNA as a gene detection reagent that can be transcribed into RNA that initiates translation into a reporter protein. Column

 Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples, but the present invention is not limited to these.

 ^ M l (construction of MB-controlled luciferase expression pattern)

 Plasmid pGL3 containing the firefly luciferase gene was purchased from Promega. Forward primer containing 3 'terminal partial region complementary to the target and SD sequence (5' _d (GGT CTG AAG GTT TAT TAG AAG GAG ATA TAC CAA TGG AAG ACG CCA AAA ACA TA) -3 ,: SEQ ID NO: 2 ) 5 pmol and reverse primer (5'_d (TATTCTATTACAC GGC GATCTTTCC G) -3 ': SEQ ID NO: 3) Using a 25 μ1 reaction mixture containing 5 pmol, the plasmid pGL3 was used as a saddle. The first PCR was performed. The first round of PCR included 5 ng of pGL3 vector, 1.25 U of Pfu Ultra HF DNA polymerase (Stratagene), 5 nmol of each dNTPs (Toyobo), and 2.5 μL of 10 X Pfu Ultra HF reaction. A buffer was used. PCR conditions were 95 ° C for 3 minutes (95 ° C for 45 seconds, 54 ° C for 45 seconds, 72 ° C for 1 minute) for 30 cycles and 72 ° C for 10 minutes.

[0098] Universal primer 5 containing a 5 'terminal partial region complementary to the target and T7 promoter, -d (GAA ATT AAT ACG ACT CAC TAT AGG GAG ACC ACA ACG GTT TCC CTC TAT CTC CTG GTC TGA AGG TTT A) A second PCR was performed using 20 μl of the reaction mixture containing 5 pmol of _3 ′ (SEQ ID NO: 4) as a double-stranded DNA generated in the first PCR reaction. In the second PCR, 5 pmol reverse primer (5, -d (TAT TCA TTA CAC GGC GAT CTT TCC G) -3, SEQ ID NO: 5), 1 / i L diluted 1/100 times the first round The same PCR reaction solution as PCR of 1.25 U of Pfu Ultra HF DNA polymerase (Stratagene), 5 nmol of each dNTPs (Toyobo), and 2.5 μL of lO X Pfu Ultra HF reaction buffer . PCR conditions were 95 ° C for 3 minutes (95 ° C for 45 seconds, 54 ° C for 45 seconds, 72 ° C for 1 minute) for 30 cycles and 72 ° C for 10 minutes.

[0099] This resulted in double-stranded DNA for MB-controlled luciferase mRNA transcription. The base sequence of this DNA is shown in SEQ ID NO: 6. In SEQ ID NO: 6, base numbers 1 to 22 are T7-promoter regions, and 6 bases of base numbers 73 to 77 are SD sequences (ribosomes). A luciferase gene whose base numbers 86 to 1722 start from the start codon. Base numbers 54 to 69 are sequences that give a complementary sequence to CCR5, which is the target sequence, when transcribed into mRNA.

 [0100] From this double-stranded DNA, MB-controlled luciferase mRNA force was obtained by ST7 run-off transcription using the T7 MEGAshortscript kit (Ambion). Briefly, Τ7 transcription reaction mixture (10 μL) containing 3 μL of the second PCR solution of vertical DNA was incubated at 37 ° C for 120 minutes. 1 U DNase I was added to the reaction mixture and the mixture was incubated for an additional 15 minutes.

 [0101] Next, mRNA was purified using the RNeasy MinElute Cleanup kit (Qiagen). The concentration of mRNA was determined by measuring the absorbance at 260 nm.

 [0102] The structure of the thus obtained MB-luciferase mRNA is shown in FIG. This RNA has a reporter gene corresponding portion and a regulatory hairpin structure upstream thereof. The hairpin structure has an 8 base long stem and a 19 base long loop. The stem contains 6 bases of RBS. The 18 bases including the loop are complementary to the 620th to 635th base sequences of the human CC chemokine receptor 5 (CCR5) gene (5, -AATAAACCTTCAGACC-3 ': SEQ ID NO: 7).

 CCR5 is an HIV-1 core receptor, and its sequence diversity is considered a haplotype marker for promoting / delaying HIV-1 infection. Therefore, the selectivity of this sequence is important.

 ^ i Luciferase translation and chemiluminescence analysis)

 Reconstructed E. coli translation solution (T7) containing 1.8 pmol of the truncated MB-luciferase mRNA obtained in Example 1 in the presence or absence of the target CCR5 oligodeoxynucleotide (18 pmol) —Transcription / translation coupled, Pure System Classic 1, Post Genome Laboratories). A total volume of 10 μL of the reaction mixture was incubated at 37 ° C. for 60 minutes.

[0103] As the target nucleic acid, an oligonucleotide (5'-CCGTAAATAAACCTTCAGACCAGAGA-3 ': SEQ ID NO: 8) containing the 620th to 635th base sequences of the CCR5 gene was used as a full-matched oligonucleotide. In SEQ ID NO: 8, Those obtained by mutating C of base number 13 to T by 1 base and those obtained by mutating C of base number 13 by 1 base to A were used as 1-base mismatched oligonucleotides, respectively. Further, a C-mutated C at base number 13 and a T-mutated base T at base number 14 were used as a 2-base mismatched oligonucleotide.

 [0104] After translation of each sample, 2.5 μL was mixed with 100 μL of luciferase Atsey reagent (Promega). 102. was transferred to a 96-well plate and the chemiluminescence intensity was measured using the Wallacl420 system.

 [0105] The results are shown in FIG. FIG. 2 is a graph showing the chemiluminescence intensity in the presence of a fully-matched oligodeoxynucleotide and a mismatched oligodeoxynucleotide as a relative value to the chemiluminescence intensity in the absence of these targets.

 Compared to the absence of target, the presence of full-matched oligonucleotides is about

Three times the emission intensity was observed. In addition, in the presence of a single-base mismatched oligonucleotide that was mutated from C to T, a luminescence intensity approximately twice that in the absence of the target was observed.

. Also mutated from C to A in the presence of 2-base mismatched oligodeoxynucleotides

In the presence of a single-base mismatched oligonucleotide, no difference was observed from the emission intensity in the absence of the target.

 Example 3 (Transcription / translation of chemoluminescence and chemiluminescence analysis)

 1. The same procedure as in Example 2 was performed, except that 0.2 pmol of MB-luciferase-expressing dsDNA cage was used in place of 8 pmol of MB-luciferase mRNA.

 [0106] Partial distribution of CCR5 gene in the same manner as in Example 2 except that 0.2 pmol of MB-luciferase expression dsDNA was used instead of 1 · 8 pmol of vertical MB-luciferase mRNA 歹 IJDNA and full-match oligo It was reacted with deoxynucleotides and three mismatched oligodeoxynucleotides.

The results are shown in FIG. About 8 times higher luminescence intensity was observed in the presence of flumati oligodeoxy tantalate than in the absence of target. In addition, even in the presence of a 1-base mismatched oligodeoxynucleotide mutated from C to T, an emission intensity about 5 times that in the absence of the target was observed. In addition, in the presence of a 2-base mismatch oligonucleotide and in the presence of a 1-base mismatch oligonucleotide mutated from C to A, About twice the emission intensity was observed compared to the absence of one get.

[0108] It can be seen that gene mutations can be detected with higher sensitivity, and differences in single bases can be detected more reliably than when RNA is used as a gene detection reagent.

[0109] Fig. 4 shows 102.5 μL of each reaction mixture transferred to a 250 μL Eppendorf tube and its chemiluminescence image measured by Versadoc3000. Figure 4 shows that even a single base mutation in a gene can be clearly identified by visual inspection.

 Confirmation of interaction with target nucleic acid sequence at translation stage)

 Cell-free T7_transcription / translation without reconstituted ribosomes containing 0.2 pmol of truncated MB-luciferase DNA and tRNA in the presence or absence of CCR5 full-matched oligonucleotide (18 pmol) In Vitro transcription was performed in Pure System Classic 1 (Post Genome Laboratories). Specifically, a total volume of 10 μL of the reaction mixture was incubated at 37 ° C for 60 minutes. 18 pmol of CCR5 full-match oligodeoxynucleotide was used. The efficiency of the transcription reaction was determined by using a diluted mRNA solution (0, 20, 40, 60, 80, 100%) of transcription mRNA in the presence of CCR5 full-matched oligonucleotide as an internal standard. This was done by comparing the amount of transcription in the presence.

 [0110] Fig. 5 shows the chemiluminescence images of each reaction mixture measured by ATTO densitograph. In the presence of the full-matched oligonucleotide, the same emission intensity as that in the absence of the target nucleotide was observed.

 [0111] From this fact, it can be seen that the translational inhibition by the target cannot be detected substantially only by the transcription stage.

 15 (Target 俘 J

In Example 2, chemiluminescence intensity was measured by changing the amount of CCR5 full-matched oligonucleotides used as targets at 0-22 pmo (FIG. 6). At about 7 pmol of CCR5 full-matched oligonucleotide, the chemiluminescence intensity is slightly saturated, but at lower concentrations, the target amount and luminescence intensity are almost proportional. In view of this force and linearity, it can be seen that the present luciferase expression type can be suitably used for detection and quantification of the target nucleic acid sequence. Example 6 (Combined use of DNA degrading enzymes)

 <Construction of MB-controlled luciferase expression type>

 The following were used as PCR primers, and double-stranded DNA for MB-controlled luciferase mRNA transcription was obtained in the same manner as in Example 1 except that the plasmid pBESTluc (Promega) was in the form of a cage. The luciferase mRNA transcribed from this double-stranded DNA is complementary to the 620th to 637th base sequences of CCR5 (5'-AATAAACCTTCAGACCAG-3 ': SEQ ID NO: 9) as a region capable of hybridizing with the target nucleic acid sequence. Have Others are the same as the double-stranded DNA strength obtained in Example 1 and the luciferase mRNA obtained.

 1st PCR

 Forward primer (5, _d (GGT CTG AAG GTT TAT TAG AAG GAG ATA TAC

CAA TGG AAG ACG CCA AAA ACA TA) _3 ': SEQ ID NO: 2)

 Reverse primer (5'—d (TAT TCA TTA CAA TTT GGA CTT TCC GCC) —3: 酉 己 column number 10)

 2nd PCR

 Universal primer (5, -d (GAA ATT AAT ACG ACT CAC TAT AGG GAG A CC ACA ACG GTT TCC CTC TAT CTC CTG GTC TGA AGG TTT A) -3 ': 酉 己 歹 lj number 4)

 Reverse primer (5, — d (TAT TCA TTA CAA TTT GGA CTT TCC GCC) — 3, 酉 己 column number 10)

 The 1 · 8 pmol MB-luciferase mRNA thus obtained was reconstituted in the presence or absence of the target CCR5 oligodeoxynucleotide (1.8 pmol) in E. coli translation solution ( T7—Transcription / Translation Coupled, Pure System Classic 1, Post Genome Laboratories). The reaction mixture with a total volume of 10 μL was incubated at 37 ° C. for 60 minutes.

[0112] Further, the same operation as described above was performed using a reaction mixture containing Tth RNaseH (0.1 U; Toyobo Co., Ltd.).

[0113] The target nucleic acid is a DNA having the 620th to 635th base sequences of the CCR5 gene. Ligonucleotide (5, -AATAAACCTTCAGACC-3 ′: SEQ ID NO: 1 1) was used as a full-match oligodeoxynucleotide. Further, in SEQ ID NO: 11, the one obtained by mutating C of base number 8 to T by 1 base was used as a 1 base mismatch oligodeoxynucleotide.

 After translation of each sample, 2.5 μL was mixed with 100 μL of Luciferase Atsey Reagent (Promega). 102. was transferred to a 96-well plate and the chemiluminescence intensity was measured using the Wallac l400 system.

[0114] The results are shown in FIG. Figure 7 shows the chemiluminescence intensity in the presence of full-matched oligonucleotides and single-base mismatched oligonucleotides, relative to the chemiluminescence intensity in the absence of target and RNaseH. It is a represented graph. Lanes! To 3 are the results in the absence of RNaseH, and lanes 4 to 6 are the results in the presence of RNaseH.

 [0115] The single base mismatch used here is due to GT base pairs, which are relatively stable "fluctuating base pairs". Since the region complementary to the target nucleotide sequence in the truncated MB-luciferase mRNA is as short as 16 mer, it is difficult to distinguish between full match oligonucleotides and GT mismatch oligonucleotides without using RNaseH (lane 2, 3) By using RNa seH, it is clear that full-matched oligonucleotides and GT-mismatched oligonucleotides can be clearly distinguished (lanes 5 and 6). Since GT1 base mismatch can be detected, other single-base mismatches can be reliably detected.

 ^ I (when the target nucleic acid is very small)

 <Translation of luciferase: Chemiluminescence analysis>

A reconstructed E. coli translation solution (T7-transcription / trans) containing 0.45 pmol of the above-mentioned type MB-luciferase mRNA in the presence or absence of the target CCR5 oligodeoxynucleotide (45 fmol). In-vitro translation was performed in the Ryge Cup Noredo, Pure System Classic 1, Post Genome Laboratories). A total reaction volume of 10 μl was incubated at 37 ° C. for 60 minutes. The number of moles of target oligonucleotide used is 1/1000 of the number of moles of vertical MB-luciferase mRNA. [0116] The same operation as described above was performed using a reaction mixture containing Tth RNaseH (lU; Toyobo Co., Ltd.).

 [0117] As the target nucleic acid, an oligonucleotide (5'-CCGTAAATAAACCTTCAGACCAGAGA-3 ': SEQ ID NO: 8) containing the 620th to 637th nucleotide sequences of the CCR5 gene was used as a full-matched oligonucleotide.

 After translation of each sample, 2.5 μL was mixed with 100 μL of Luciferase Atsey Reagent (Promega). 102. was transferred to a 96-well plate and the chemical emission intensity was measured using Lumat LB9507.

 [0118] The results are shown in FIG. Lanes 1 and 2 are the results in the presence of RNaseH, and lanes 3 and 4 are the results in the absence of RNaseH. Even when the number of targets was very small, by using RNase e, very strong chemiluminescence was observed compared to the case where no target was present. It can be seen that transcription and translation of noreciferase were detected with a very small amount of target.

 Example 8 (Detection using β-galactosidase)

 <Construction of MB-regulated 8-galactosidase expression type>

 MB-controlled dsDNA for 3-galactosidase mRNA transcription was obtained in the same manner as Example 1 except that the following PCR primers were used. The β-galactosidase mRNA transcribed from this double-stranded DNA has an mRNA encoding i3-galactosidase in place of the mRNA encoding luciferase. Further, as a region that can be hybridized with the target nucleic acid sequence, it has a sequence complementary to the 620th to 637th base sequences of CCR5 (5′—AATAA ACCTTCAGACCAG—3 ′: SEQ ID NO: 9). Others are the same as the double-stranded DNA strength obtained in Example 1 and the obtained luciferase mRNA.

 1st PCR

 Forward primer (5, _d (GGT CTG AAG GTT TAT TAG AAG GAG ATA TAC CAA TGA CCA TGA TTA CGG ATT CAC) -3 ': SEQ ID NO: 12)

 Reverse primer (5'_d (GGA TTA GTT ATT CAT TAT TTT TGA CAC CAG AC C AAO-3 ': SEQ ID NO: 13)

2nd PCR Forward primer (5, -d (GAA ATT AAT ACG ACT CAC TAT AGG GAG ACC ACA ACG GTT TCC CTC TAT CTC CTG GTC TGA AGG TTT ATT AG) -3, sequence number 14)

 Reverse primer (5, _d (GGA TTA GTT ATT CAT TAT TTT TGA CAC CAG A

CC AAO-3 ': SEQ ID NO: 13)

 <Translation of R-galactosidase ^ color>

 Reconstructed E. coli translation solution containing 0.2 pmol of MB-β-galatatosidase dsDNA in the presence or absence of target CCR5 oligodeoxynucleotide (18 pmol) (T7_transcription / Translations were performed in Translation Coupled, Pure System Classic 1, Post Genome Laboratories. A total volume of 10 μL of the reaction mixture was incubated at 37 ° C. for 60 minutes.

[0119] Further, the same operation as described above was performed using a reaction mixture containing Tth RNaseHdU (Toyobo Co., Ltd.).

 [0120] As the target nucleic acid, an oligonucleotide (5'-CCGTAAATAAACCTTCAGACCAGAGA-3 ': SEQ ID NO: 8) containing the 620th to 637th base sequences of the CCR5 gene was used as a full-matched oligonucleotide.

 [0121] After translating each sample, i3-galactosidase activity was measured using the galactosidase enzyme assembly system (Promega) according to the attached protocol. However, 1 1 / i L of the translation solution was mixed with 39 μL of 1 X reporter lysis buffer and 50 μL of Atsy 2 X buffer containing οnitrofenenore β D galactopyranoside (ONPG).

 [0122] The results are shown in Fig. 9 (Α). Lanes 1 and 2 are the results in the absence of RNaseH, and lanes 3 and 4 are the results in the presence of RNaseH. In the presence of a full-match target, a stronger absorption at 405 nm due to the formation of nitrophenol was observed compared to the absence of the target. In addition, the use of RNaseH made it clearer from the case where no target was present.

Figure 9 (B) shows the color development by β-galatatosidase. 2 and 3 are when RNaseH is used, 2 is when the target is not present, and 3 is when the full-match target is present. 4 and 5 are when RNaseH is not used, and 4 is the target If not, 5 is when a full-match target is present. 1 is the same as 4. 1 and 2 were transparent, but 3 was strongly yellow. 4 was transparent but 5 was colored yellow. In other words, strong color development was observed due to the presence of the full-match target compared to the case where the target did not exist. In addition, the use of RNaseH made it more distinct from the case where no target was present. Since the reaction product nitrophenol absorbs in the visible region, the target can be detected visually without using a special device if the j3_galactosidase gene is used as the reporter gene.

Claims

The scope of the claims  [1] A gene detection reagent comprising a region capable of hybridizing with a target nucleic acid sequence, a ribosome binding region, an initiation codon, and a region encoding a reporter protein, and a higher-order structural change is induced by hybridization with the target nucleic acid. A gene detection reagent that is translated into a reporter protein.  [2] The gene detection reagent according to claim 1, which has at least a region strength NA encoding a ribosome binding region, an initiation codon, and a reporter protein. [3] The ribosome cannot bind to the ribosome binding region, and the ribosome can bind to the higher-order structural change cabsome binding region induced by hybridization between the target nucleic acid and the target nucleic acid. The gene detection reagent according to claim 2, which initiates translation into a reporter protein. [4] A region comprising a ribosome binding region, an initiation codon, and a region encoding a reporter protein in order from upstream, and a region including the ribosome binding region further upstream of the ribosome binding region via a region capable of hybridizing with the target nucleic acid sequence. 4. The gene detection reagent according to claim 3, comprising a region capable of hybridizing with the gene. [5] The gene detection reagent according to claim 4, wherein the ratio of the number of bases in the region hybridizable to the target nucleic acid sequence to the number of bases in the region hybridizable to the region including the ribosome binding region is 1 to 10: [6] The gene detection reagent according to claim 4, wherein the region capable of hybridizing with a region including a ribosome binding region comprises 5 to 30 bases, and the region capable of hybridizing with a target nucleic acid sequence comprises 5 to 60 bases.  [7] The reagent for gene detection according to claim 1, wherein the region capable of hybridizing with the target nucleic acid sequence is a region complementary to the target nucleic acid sequence. [8] The gene detection reagent according to claim 1, comprising a region that functions as a ribozyme that self-cleaves when hybridized with a target nucleic acid. [9] The reagent for gene detection according to claim 1, wherein the reporter protein is an enzyme. 10. The gene detection reagent according to claim 9, wherein the enzyme is involved in the production of a substance that generates visible light. [11] Equipped with a region that can hybridize with the target nucleic acid sequence, a ribosome binding region, an initiation codon, and a region encoding the reporter protein, and higher-order structural changes induced by hybridization with the target nucleic acid initiate translation into the reporter protein Reagent for gene detection containing DNA that can be transcribed into RNA.  [12] The RNA is in a state where the ribosome cannot bind to the ribosome binding region, and the conformational change induced by hybridization between the target nucleic acid and the target nucleic acid can bind the ribosome to the ribosome binding region. 12. The gene detection reagent according to claim 11, which initiates translation into a reporter protein.  [13] The RNA comprises a region encoding a ribosome binding region, an initiation codon, and a reporter protein in order from upstream, and further upstream of the ribosome binding region via a region capable of hybridizing with a target nucleic acid sequence. 13. The gene detection reagent according to claim 12, comprising a region capable of hybridizing with a region containing a binding region.  [14] The reagent for gene detection according to claim 13, wherein the ratio of the number of bases in the region hybridizable with the target nucleic acid sequence to the number of bases in the region hybridizable with the region including the ribosome binding region is 1 to 10:  15. The gene detection reagent according to claim 13, wherein the region capable of hybridizing with a region containing a ribosome binding region comprises 5 to 30 bases, and the region capable of hybridizing with a target nucleic acid sequence comprises 5 to 60 bases.  [16] The gene detection reagent according to claim 11, wherein the region capable of hybridizing with the target nucleic acid sequence is a region complementary to the target nucleic acid sequence. [17] The reagent for gene detection according to [11], wherein the RNA includes a region that functions as a ribozyme that self-cleaves when hybridized with a target nucleic acid. 18. The gene detection reagent according to claim 11, wherein the reporter protein is an enzyme. [19] The gene detection reagent according to [18], wherein the enzyme is involved in the production of a substance that generates visible light. [20] A gene detection kit comprising the gene detection reagent according to claim 1. 21. The gene detection kit according to claim 20, further comprising a prokaryotic cell extract. [22] In addition, an RNase that specifically cleaves RNA hybridized with DNA is provided. The gene detection kit according to claim 20.  [23] A gene detection kit comprising the gene detection reagent according to claim 11.  24. The gene detection kit according to claim 23, further comprising a prokaryotic cell extract.  25. The gene detection kit according to claim 23, further comprising an RNase that specifically cleaves RNA hybridized with DNA.  [26] The first step of mixing the test sample and the gene detection reagent according to claim 1, and the expression level of the reporter protein from the gene detection reagent in the absence of the test sample. And a second step of determining the presence or absence of the target nucleic acid in the test sample by comparing the expression level of the reporter protein from the gene detection reagent described in 1.  [27] The method according to claim 26, wherein the expression of the reporter protein from the gene detection reagent is carried out in the presence of a prokaryotic cell extract.  [28] The first step includes a step of mixing the test sample, the gene detection reagent according to claim 1, and an RNase that specifically cleaves the hybridized RNA with DNA. The method described.  [29] The first step of mixing the test sample with the gene detection reagent according to claim 11, and the expression level of the reporter protein from the gene detection reagent according to claim 11 in the absence of the test sample. A gene detection method comprising a second step of determining the presence or absence of a target nucleic acid in a test sample by comparing with the expression level of a reporter protein from the gene detection reagent described.  [30] The method of claim 29, wherein the reporter protein is expressed from the gene detection reagent in the presence of a prokaryotic cell extract.  [31] The first step includes the step of mixing the test sample, the gene detection reagent according to claim 1, and an RNase that specifically cleaves the hybridized RNA with DNA. The method described. [32] A first step of mixing a test sample, the gene detection reagent according to claim 1 with an RNase that specifically cleaves RNA hybridized with DNA, and a gene detection reagent By comparing the expression level of the reporter protein in the absence of the test sample with the expression level of the reporter protein from the reagent for gene detection according to claim 1. A method for detecting a single nucleotide polymorphism comprising a second step of determining the presence or absence of a target nucleic acid in a test sample.  [33] A test sample, the gene detection reagent according to claim 11, and R hybridized with DNA The gene according to claim 11, wherein the first step of mixing with an RNase that specifically cleaves NA and the expression level of the reporter protein in the absence of the test sample are determined using the reagent for detecting the gene and the expression level of the reporter protein. A single nucleotide polymorphism detection method comprising a second step of determining the presence or absence of a target nucleic acid in a test sample by comparing with the expression level of a reporter protein from a detection reagent.  [34] A nucleic acid comprising a region capable of hybridizing with a target nucleic acid sequence, a ribosome binding region, an initiation codon, and a region encoding a reporter protein, wherein a conformation change is induced by hybridization with the target nucleic acid, and the reporter Use of a nucleic acid that begins translation into a protein as a gene detection reagent. [35] RNA comprising a region capable of hybridizing with a target nucleic acid sequence, a ribosome binding region, an initiation codon, and a region encoding a reporter protein, and a conformational change induced by hybridization with the target nucleic acid to the reporter protein Use as a gene detection reagent for DNA that can be transcribed into RNA that initiates translation of RNA.