JP2005110664A - Method for detecting presence of target dna by amplification of nucleic acid signal, and method for detecting or determining immunological ligand - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting a target DNA without performing the amplification of the target DNA for performing the detection of the target DNA and without being limited by the base sequence of the target DNA. <P>SOLUTION: This method for detecting the presence of the target DNA is provided by using a target DNA-detecting probe obtained by connecting a complemental base sequence to the target DNA and an optionally selectable base sequence which is not complemental, and amplifying a nucleic acid signal composed of oligo nucleotide containing a base sequence complemental to the base sequence which is not complemental to the target DNA in the probe. Although the base sequence of the target DNA is different, the amplified products can be detected by the probe having the same base sequence. The method for detecting the presence of the target DNA can be applied to the detection or determination of an immunological ligand. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for detecting the presence of a target DNA by amplifying a nucleic acid signal which can be arbitrarily set without amplification of the target DNA itself and comprises a preset oligonucleotide. Furthermore, the present invention relates to a method for detecting or quantifying an immunological ligand by applying the method for detecting the presence of the target DNA.

  Deoxyribonucleic acid (DNA) is a nucleic acid in which a large number of deoxyribonucleotides are polymerized, and deoxyribonucleotides are linearly linked to form a genetic code. DNA is the main body of a gene, and the gene exists in the state of double-stranded DNA in which single-stranded DNAs composed of complementary base sequences are hybridized.

  Many DNA amplification techniques have been developed so far and are widely used as tools for detecting and analyzing extremely small amounts of DNA. Genetic testing for detecting a specific base sequence and amplification of a specific base sequence for analyzing a small amount of DNA are becoming indispensable technologies due to increasing demand in research, clinical and industrial fields. ing. The polymerase chain reaction (PCR) is known as the most widely used DNA amplification method (Non-patent Document 1).

  PCR uses two types of primers designed to hybridize to the target DNA and its complementary strand. The primer and target DNA are hybridized by increasing and decreasing the temperature. The 3 'end of the hybridized primer serves as a replication start point, and a polymerase reaction proceeds by adding deoxyribonucleotides using the target DNA as a template in the presence of DNA polymerase. When the strand synthesized in this way is dissociated from the target DNA by increasing the temperature, and further hybridized with the other primer by decreasing the temperature, the same reaction is performed using the synthesized strand as a template. proceed. Thus, by repeating the temperature change, the target DNA can be amplified as a double strand.

  Similarly, ligase chain reaction (LCR) has been put to practical use as a method for amplifying a target DNA that requires repeated temperature changes, and details are described in Patent Document 1. LCR is a method in which two probe pairs designed to hybridize to a target DNA are prepared, and a new strand is synthesized by linking two probes hybridized to the target DNA. Since PCR and LCR require temperature change, special equipment is required, and since one cycle of temperature change requires several minutes, there are problems that the reaction time becomes longer when the number of cycles is large. .

  In order to improve the above-mentioned problems, isothermal DNA amplification techniques that do not require temperature changes have been developed. The strand displacement amplification method (SDA method) is designed so that a restriction enzyme recognition sequence is added to the primer 5 ′ side that hybridizes to the target DNA, and the downstream base sequence hybridizes with the target DNA (non-patent document). 2). When the primer and target DNA are hybridized, the 3 'end of the primer becomes the replication start point, and the polymerase reaction proceeds using the target DNA as a template. At the same time, the 3' end of the target DNA becomes the replication start point and the restriction enzyme recognition sequence of the primer is used as a template. As the polymerase reaction proceeds. At that time, in order to avoid the cleavage of the strand on the target DNA side by the restriction enzyme, a modified nucleotide is incorporated at a specific position. When a restriction enzyme acts on the double-stranded DNA thus formed, only the strand containing the primer is cleaved (nicked), and the 3 ′ end is formed. The polymerase reaction proceeds. If primers that hybridize to the complementary strand of the target DNA are simultaneously added to the reaction system, the target DNA is amplified in a double-stranded state isothermally.

  However, in the SDA method, hybridization between the primer and the target DNA occurs downstream of the restriction enzyme recognition sequence of the primer, and the 3 ′ end of the target DNA becomes the replication origin and a complementary strand of the restriction enzyme recognition sequence of the primer is synthesized. In order to achieve this, it is necessary to fragment the target DNA in advance. An improved SDA method that eliminates such trouble has been developed (Patent Document 2), but in this case, it is necessary to design four types of primers. In the SDA method, since nicking is caused by a restriction enzyme, an appropriate position of the restriction enzyme recognition sequence is substituted with a phosphorothioate bond (so-called S-bond). The strand that has been converted to S by this modification is avoided from cleavage by restriction enzymes, and only its complementary strand is cleaved. In the SDA method, the reaction temperature can be advanced in the range from about 37 ° C. to about 65 ° C. depending on the enzyme used, but it is 60 ° C. in order to avoid non-specific interaction between primers and increase the reaction rate. It is considered desirable to react at a high temperature in the vicinity. Patent Document 3 describes in detail the position of S-formation necessary for nicking a thermostable strand displacement polymerase, restriction enzyme, and restriction enzyme recognition sequence that can be used in a reaction at high temperature. According to it, the formation of nicking is not possible with all restriction enzymes and the types of restriction enzymes are limited.

  Loop-mediated Isometric Amplification (Lamp method) is a DNA amplification method in which self-amplification is possible when the first amplification product has a loop structure (Non-patent Document 3). However, four types of specially structured primers are required, and the amplification product is an inconsistent size of DNA in which the region targeted for amplification is repeated.

  In the “Isothermal and Chimeric Primer-Initiated Amplification of Nucleic Acids” (ICAN method), a chimeric primer having a DNA structure on the 5 ′ side and an RNA structure on the 3 ′ side is used (Patent Document 4). When the primer and the target DNA are hybridized, the primer 3 ′ end becomes the replication start point, and the polymerase reaction proceeds using the target DNA as a template. At the same time, the RNase H added in the reaction solution hybridizes with the target DNA in the primer sequence. A part of the RNA structure in soybean is cleaved. The 3 'end of the primer sequence thus generated becomes a replication start point, and the complementary strand of the target DNA is continuously synthesized. If primers that hybridize to the complementary strand of the target DNA are simultaneously added to the reaction system, the target DNA is amplified in a double-stranded state isothermally. However, synthesis of chimeric primers consisting of DNA and RNA is generally not widely used, and furthermore, there is a risk that the RNA portion will be easily decomposed if care is not taken. Has not reached.

  Each of the DNA amplification methods shown above is a method for amplifying the base sequence of the target DNA itself. Therefore, the sequence of the primer or probe is strongly regulated by the base sequence of the target DNA, and it is often difficult to select a combination of sequences in which non-specific amplification hardly occurs. DNA probes are often used to detect amplification products. This is a method for detecting an amplification product by hybridizing it to a DNA probe having a base sequence complementary to the amplification product, and is a very effective means for specifically detecting the amplification product.

  However, in the DNA amplification method shown above, the probe sequence used to detect the amplification product is also restricted by the base sequence of the target DNA, and at the same time, at least one type of target DNA among several types of target DNA is present. Even in a screening test or the like that wants to know the existence, it is necessary to prepare the same number of DNA probes as the type of the target DNA for detecting the amplification product. In order to amplify and analyze a specific base sequence, it is necessary to amplify the base sequence of the target DNA as it is, but the purpose of DNA amplification technology in clinical and industrial fields is mostly the presence of the target DNA. It is to determine the presence or absence of. For example, in the clinical field, DNA amplification technology is used as a means to determine the presence of viruses and bacteria that cause disease in patients based on the presence of genes, and to investigate the presence of genes related to various diseases. DNA amplification technology is used as a means for determining the presence of food poisoning bacteria in the presence of genes.

  Patent Document 5 describes an in situ synthesis method of oligonucleotides at isothermal conditions. According to this, it is possible to nick a complementary strand by modifying an oligonucleotide that serves as a template or a primer, and a single-stranded oligonucleotide can be synthesized continuously. However, the method described in this document cannot be designed to be an arbitrary base sequence suitable for detection because the base sequence to be amplified is limited to the target DNA portion.

European Patent Application Publication No. 0320308 Japanese Patent Publication No. 8-76 JP-A-7-289298 WO00 / 56877 brochure WO92 / 05287 pamphlet Science, 230, 1350-1354, 1985 Proc. Natl. Acad. Sci. USA, 89, 392-396; 1992. Nucleic Acids Research, 2000, Vol. 28, No. 12

  An object of the present invention is to provide a method for detecting a target DNA that is not regulated by the base sequence of the target DNA without performing amplification of the target DNA in order to detect the target DNA. An object of the present invention is to provide a method for detecting a target DNA in which all amplification products can be detected with a probe having the same base sequence even if the base sequences are different. It is intended to provide a method for detecting a target DNA by determining the presence or absence of DNA as a DNA having an arbitrary length and sequence that is not regulated by the base sequence of the target DNA. Furthermore, another object of the present invention is to provide a method for detecting or quantifying immunological ligands by applying a method for detecting the presence of target DNA.

  If a method for detecting a nucleic acid signal comprising an oligonucleotide having an arbitrary base sequence amplified by the presence of the target DNA can be realized rather than a method for amplifying and detecting the base sequence of the target DNA itself, the detection method can be The present inventors paid attention to the fact that they become more efficient and have a wide range of applications, and found a method capable of detecting the target DNA itself without amplifying it in order to detect the target DNA.

  That is, the method for detecting the presence of the target DNA of the present invention uses a target DNA detection probe in which a base sequence complementary to the target DNA and an arbitrarily selectable base sequence that is not complementary are bound, A nucleic acid signal comprising an oligonucleotide containing a base sequence complementary to a base sequence that is not complementary to the target DNA in the target DNA detection probe is amplified.

  In the method for detecting the presence of the target DNA of the present invention, the target DNA detection probe to be used has a base sequence complementary to the base sequence of one strand of the double-stranded target DNA at the 3 ′ side, and The 5′-side portion is characterized by an arbitrarily selectable base sequence that is not complementary to the target DNA. Furthermore, in the method for detecting the presence of the target DNA of the present invention, the target DNA detection probe to be used has a base sequence portion capable of hybridizing with a restriction enzyme recognition sequence present in the target DNA, and the base sequence portion. Includes at least one base sequence modified so as to be protected from cleavage by a restriction enzyme.

A method for detecting the presence of a desired more specific target DNA of the present invention comprises:
1) The 3 ′ side portion is a base sequence complementary to one strand of a double-stranded target DNA, and the 5 ′ side is not complementary to the target DNA, and an arbitrarily selectable base sequence (5 ′ arm sequence and And the target DNA detection probe has a base sequence portion capable of hybridizing with a restriction enzyme recognition sequence present in the target DNA, and the base sequence portion. Provides a target DNA detection probe comprising at least one base sequence modified so as to be protected from cleavage by a restriction enzyme;
2) hybridizing the target DNA detection probe and the target DNA;
3) Let the extension reaction of the 3 ′ end of the hybridized target DNA detection probe obtained in the step 2) be performed,
4) Nicking is performed by allowing a restriction enzyme to act on the restriction enzyme recognition site of the hybridized target DNA obtained in step 3).
5) An extension reaction of the 3 ′ end of the target DNA is performed by a strand displacement DNA polymerase using the 3 ′ end of the cleavage site of the target DNA generated by the nicking obtained in the step 4) as a replication start point. 'Create a double strand consisting of a complementary strand to the target DNA detection probe containing the arm sequence,
6) Cleavage of the target DNA by nicking by causing a restriction enzyme to act on the double-stranded restriction enzyme recognition site obtained in the step 5),
7) An extension reaction of the 3 ′ end of the target DNA is carried out by a strand displacement DNA polymerase using the 3 ′ end of the cleavage site of the target DNA generated by the nicking obtained in the step 6) as a replication start point. 'A double-stranded DNA comprising a complementary strand to a target DNA detection probe containing an arm sequence is prepared, and at the same time, an oligonucleotide containing a base sequence complementary to the 5' arm sequence is released.
8) A nucleic acid signal composed of an oligonucleotide containing a base sequence complementary to the 5 ′ arm sequence is amplified by repeating the steps 6) and 7).

  1. According to the method for detecting the presence of the target DNA of the present invention, since the target DNA itself is not amplified, a method for detecting a target DNA that is not regulated by the base sequence of the target DNA can be provided.

  2. In addition, according to the method for detecting the presence of the target DNA of the present invention, even if the base sequences of the target DNA are different, all amplification products can be detected with probes having the same base sequence, which is useful for screening tests. is there.

  That is, in the conventional method of amplifying the base sequence of the target DNA itself and detecting it with the probe, the base sequence of the probe needs to have a base sequence complementary to the base sequence of the target DNA. When amplifying and detecting, it is necessary to prepare as many probe types as the number of target DNA types, and at the same time, hybridization between the amplification products and the probes must be performed in a separate reaction system for each amplification product. There is.

  On the other hand, according to the present invention, even if the base sequence of the target DNA is different, the amplification product can be amplified as an oligonucleotide containing the same base sequence. Therefore, several types of target DNA can be detected in the same reaction system with a single type of probe. For example, it is effective when detecting the presence (screening test) of at least one of several types of target DNA.

  Furthermore, when preparing a test agent used in the method for detecting the presence of the target DNA of the invention, if one kind of probe for detecting the amplification product is prepared, it can be applied to detection of any target DNA. The labor of reagent development, the process of the detection method of the present invention are simplified, and there are advantages in terms of cost.

  3. In addition, according to the method for detecting the presence of the target DNA of the present invention, target DNA detection in which a base sequence complementary to the target DNA and an arbitrarily selectable base sequence that is not complementary to the target DNA are bound to each other is detected. Therefore, a target DNA detection probe having a base sequence suitable for detection can be designed.

  That is, it is known that DNA hybridization is greatly influenced by the base composition, such as binding strength and non-specificity. For example, when designing primers for PCR, simply designing primers with sequences at both ends of the region that you want to increase will not be amplified by PCR, the amplification rate is extremely low, or nonspecific hybridization Often appears prominently. This is because specific hybridization between the primer and the target DNA is not performed at a high rate. Therefore, it is necessary to search for a sequence suitable for the primer.

  The same applies to the hybridization between the amplification product and the probe for detecting the amplification product. When amplifying the base sequence of the target DNA itself, the base sequence of the amplification product is naturally restricted by the base sequence of the target DNA, and thus cannot be freely selected. Therefore, hybridization between an amplification product and a probe is not necessarily non-specific and does not necessarily have an appropriate binding force. For example, the amplification product or the probe itself may take an intramolecular higher order structure. The intramolecular conformation significantly reduces the hybridization rate.

  On the other hand, in the method for detecting the presence of the target DNA of the present invention, the amplification product can be made into an arbitrary free base sequence. It is possible to make the base sequence suitable for hybridization.

  4). The method for detecting the presence of target DNA using the target DNA detection probe in the present invention detects or quantifies an immunological ligand or immunological anti-ligand, specifically, an antigen or an antibody. Applicable to the method.

  The principle of the method for detecting the presence of target DNA by amplification of the nucleic acid signal of the present invention will be described below with reference to the drawings.

Reaction step I (reaction step in which double-stranded target DNA is dissociated into single-stranded target DNA by denaturation treatment):
As shown in FIG. 1, a double-stranded target DNA having a restriction enzyme recognition sequence is dissociated by denaturation treatment to produce a single-stranded target DNA. Examples of the modification treatment include heat treatment, alkali treatment, desalting treatment, formamide treatment, urea treatment and the like.

Reaction step II (reaction step of hybridizing the target DNA detection probe and the single-stranded target DNA):
A nucleotide sequence complementary to a sequence portion including a restriction enzyme recognition sequence of a single-stranded target DNA, and an arbitrary nucleotide sequence introduced on the 5 ′ side of the sequence, the nucleotide sequence of the single-stranded target DNA A target DNA detection probe comprising a base sequence that is not complementary to is prepared in advance. In the base sequence of the target DNA detection probe, a base sequence that is not complementary to the base sequence of the single-stranded target DNA is referred to as a “5 ′ arm sequence”. By introducing a phosphorothioate bond into the cleavage site of the restriction enzyme recognition sequence in the target DNA detection probe, it is prevented from being cleaved by the restriction enzyme. As shown in FIG. 2, the target DNA detection probe and the single-stranded target DNA obtained in the step I are hybridized by a temperature drop.

Reaction step III (reaction step causing nicking of target DNA by restriction enzyme):
The presence of a polymerase (strand displacement type DNA polymerase) and a restriction enzyme in the reaction product obtained in the reaction step II allows the target DNA detection probe 3 by polymerase (strand displacement type DNA polymerase) as shown in FIG. 'End extension reaction and cleaving (nicking) only the target DNA side with restriction enzymes. The order of addition of the polymerase (strand displacement DNA polymerase) and the restriction enzyme is arbitrary and may be simultaneous. Hybridization is stabilized by the extension reaction of the 3 ′ end of the target DNA detection probe by a polymerase (strand displacement type DNA polymerase). The site cleaved by the restriction enzyme is a restriction enzyme recognition site only on the target DNA side and is not cleaved because a phosphorothioate bond is introduced into the restriction enzyme recognition site on the target DNA detection probe side.

Reaction step IV (reaction step with strand displacement type DNA polymerase):
As shown in FIG. 3, the strand displacement type DNA polymerase recognizes the 3 ′ end on the target DNA side generated by the nicking obtained in the reaction step III as a replication origin, and the 5 ′ arm sequence of the target DNA detection probe is detected. The strand displacement polymerase reaction proceeds using a sequence upstream of the restriction enzyme recognition sequence as a template.

Reaction step V (nicking step on the target DNA side of the double-stranded restriction enzyme recognition sequence):
A restriction enzyme is allowed to act on the double strand formed in the reaction step IV, thereby cleaving (nicking) only the target DNA side (FIG. 4).

Reaction step VI (strand displacement type DNA polymerase reaction step):
The strand displacement DNA polymerase recognizes the 3 ′ end, which is the cleavage site of the target DNA generated in the reaction step V, as the replication origin, and is upstream of the restriction enzyme recognition sequence including the 5 ′ arm sequence of the target DNA detection probe. Reaction with strand displacement DNA polymerase proceeds using the sequence as a template (FIG. 4).

Reaction Step VII (Nucleic acid signal amplification reaction step comprising a strand-displacing DNA polymerase and an oligonucleotide complementary to the 5 ′ arm sequence by restriction enzyme):
By repeating the reaction step V and the reaction step VI alternately, a nucleic acid signal containing a complementary strand of the 5 ′ arm sequence is continuously produced and amplified (FIG. 4).

Reaction step VIII (detection of nucleic acid signal containing the complementary strand of the amplified 5 'arm sequence):
Any known nucleic acid detection method may be used to detect a nucleic acid signal containing the 5 ′ arm sequence amplified in reaction step VII. In the method for detecting the presence of the target DNA of the present invention, the produced amplification product can be obtained by adding nucleotides using the target DNA detection probe as a template. At this time, various known modified nucleotides such as biotin are used. Labeled nucleotides such as labels, fluorescent labels, DIG labels and the like can be incorporated. For example, the following detection methods 1 and 2 of a nucleic acid signal can be applied to a detection method by hybridization of an amplified nucleic acid signal and a probe.

Method for detecting nucleic acid signal by hybridization with probe 1:
This method is a method of detecting a nucleic acid signal by fluorescently labeling an amplification product by incorporating fluorescently labeled dNTP in the process of amplifying a complementary strand of a 5 ′ arm sequence. According to this method, if a fluorescently labeled dNTP is added to the reaction solution in addition to the dNTP corresponding to the four types of bases, the fluorescently labeled dNTP is incorporated at a certain ratio in the amplification product, so that the nucleic acid signal can be detected. It becomes possible.

  Another method for detecting a nucleic acid signal is to use two types of probes to detect without incorporating fluorescence. For example, two types of probes are prepared, one is a solid phase probe for trapping an amplification product and the other is a probe emitting a fluorescent signal. Since these two probes are designed to hybridize to different positions in the amplification product sequence, they can simultaneously hybridize to the amplification product. Since the amplification product can be kept in a native state, there is an advantage that there is no inconvenience that a high rate of amplification reaction is reduced and recognition of restriction enzymes is lowered.

Method 2 of detecting nucleic acid signal by hybridization with probe:
In the same manner as in Detection Method 1, biotin-labeled dNTP is incorporated into the amplification product and the amplification product is labeled with biotin. Subsequently, if an enzyme is bound to avidin or streptavidin, a biotin-avidin or biotin-streptavidin bound can be detected by an enzymatic reaction. Examples of the enzyme used for the enzyme reaction include alkaline phosphatase and peroxidase.

  The “target DNA” in the present invention means DNA when DNA to be detected is DNA, and DNA when reversely transcribed using RNA as a template when RNA to be detected is RNA. Therefore, the method for detecting the presence of the target DNA in the present invention is not limited to DNA. In the case of RNA, RNA can also be detected by previously synthesizing DNA from RNA using reverse transcriptase. is there.

  The “double-stranded DNA” in the present invention means DNA in which base sequences complementary to each other are bonded side by side by hydrogen bonding, and double-stranded DNA having an arbitrary base sequence and two encoding genes. Double-stranded DNA encoding part of the DNA and part of the gene is included.

  Furthermore, “single-stranded DNA” in the present invention refers to DNA that does not separate into two or more molecules when the base pair bond of a complementary base sequence is dissociated.

  As a complementary base sequence in the present invention, it is not necessary for all base sequences to be completely complementary, and it is sufficient if there is sufficient complementarity to hybridize at the reaction temperature.

  The target DNA detection probe used in the present invention does not necessarily need to be deoxyribonucleic acid (DNA), and at least a part of the target DNA detection probe has a specificity similar to base pair complementation. For example, peptide nucleic acid (PNA), ribonucleic acid (RNA), etc. may be sufficient. Therefore, the case where at least a part of the target DNA detection probe used in the present invention is PNA or RNA is included. In the probe for detecting a target DNA used in the present invention, nucleotides do not need to be linked by phosphodiester bonds, and all bonds capable of being linked by a polymerase (strand displacement DNA polymerase) such as phosphorothioate bonds are possible. .

  The 5 'arm sequence in the target DNA detection probe of the present invention can be composed of at least one kind of deoxyribonucleotide, and its length and sequence are arbitrary.

  The restriction enzyme recognition sequence contained in the target DNA detection probe in the present invention can be recognized by a restriction enzyme, but at least one nucleotide is modified and protected from cleavage by a restriction enzyme to form a double-stranded DNA. It contains recognition sequences for all restriction enzymes that can sometimes be nicked.

  In the present invention, “nicking” refers to protecting a single-stranded specific site in double-stranded DNA and cleaving the remaining single-stranded restriction enzyme recognition site with a restriction enzyme.

  The amplified product from the reaction step VII (referred to as “first amplified product”) has a base sequence complementary to the base sequence, which is connected to the 5 ′ side and the 3 ′ side centering on the S-restricted restriction enzyme recognition sequence. The amplification probe is hybridized to the 3 ′ side, and further, a polymerase (strand displacement DNA polymerase) reaction using the 3 ′ end of the amplified product as a replication origin and subsequent nicking with a restriction enzyme are continuously performed. As a result, the second amplification product having the same base sequence as the first amplification product is continuously produced, and the second amplification product also hybridizes to the amplification probe, so that the amplification product is amplified exponentially. become.

  Both the restriction enzyme and the strand displacement type DNA polymerase used in the present invention have heat resistance, and preferably have heat resistance capable of performing an enzymatic reaction at a high temperature of 60 ° C. Desirable to reduce production and increase reaction rate.

  The restriction enzyme recognition sequences present in the target DNA and the target DNA detection probe used in the method for detecting the presence of the target DNA of the present invention are preferably BsrSI, BsrI, BstOI, BstNI, BsmAI, BslI and BsoBI. A sequence recognized by a restriction enzyme selected from the group consisting of:

  The strand displacement DNA polymerase used in the method for detecting the presence of the target DNA of the present invention is preferably Bca or Bst.

  Hybridization of the target DNA detection probe and the target DNA in the reaction step II is 0 or 1 base or more from the 5 ′ side to the 3 ′ end of the restriction enzyme recognition sequence of the probe and is complementary to the base sequence of the target DNA. Is desirable for restriction enzymes to efficiently recognize restriction enzyme recognition sequences.

  In the above case, a probe is designed in the case where a preferable restriction enzyme recognition sequence capable of causing nicking is present in the base sequence of the target DNA, and the complementary strand of the 5 ′ arm sequence is continuously synthesized. If there is no restriction enzyme recognition site that can cause nicking in the double-stranded target DNA sequence, a nucleic acid containing a complementary strand of the 5 ′ arm sequence can be continuously synthesized by performing the treatment shown in FIG.

  That is, as shown in FIG. 5, a restriction enzyme recognition site that cannot cause nicking is searched. When a restriction enzyme recognition site that cannot cause nicking is a site cleaved by restriction enzyme A (restriction enzyme A recognition site), the double-stranded target DNA is cleaved by restriction enzyme A.

  Next, the cleaved double-stranded target DNA is denatured by heat treatment or the like to dissociate the double-stranded target DNA to obtain a single-stranded target DNA having a 3 ′ end at the cleavage site by the restriction enzyme A.

  A restriction enzyme recognition sequence different from the restriction enzyme A recognition site in the base sequence of the target DNA and not complementary to the base sequence of the target DNA (referred to as a restriction enzyme B recognition sequence) 'Set in the arm arrangement. The restriction enzyme B recognition sequence in the target DNA detection probe is not cleaved by restriction enzyme B by introducing a phosphorothioate bond into the sequence (S). The base sequence of the target DNA detection probe is, in order from the 5 ′ side, a S-modified restriction enzyme recognition sequence that is included on the 3 ′ side of the 5 ′ arm sequence and 5 ′ arm sequence and can cause nicking, and It consists of a base sequence in which the single-stranded target DNA of the step and a complementary base sequence are combined. Such a target DNA detection probe is hybridized with the single-stranded target DNA obtained in the above step.

  Next, by applying a polymerase (strand displacement type DNA polymerase), the extension reaction proceeds from the 3 ′ end of the cleavage end by the restriction enzyme A derived from the single-stranded target DNA, and the base sequence complementary to the 5 ′ arm sequence Is formed.

  Next, by making restriction enzyme B act, the restriction enzyme B recognition site contained in the base sequence complementary to the 5 'arm sequence and contained in the base sequence on the target DNA side is nicked.

  Next, the strand displacement type DNA polymerase recognizes the strand displacement type DNA polymerase by using the 3 ′ end of the restriction enzyme B cleavage site on the target DNA side as the replication origin by acting on the strand displacement type DNA polymerase, and the 5 ′ arm sequence of the target DNA detection probe The strand displacement type polymerase reaction proceeds using a sequence upstream of the restriction enzyme B recognition sequence containing as a template. By such action of the DNA polymerase and the restriction enzyme, the complementary strand cleaved from the 5 'arm sequence of the target DNA detection probe can be continuously produced.

  As the DNA polymerase used in the method for detecting the presence of the target DNA of the present invention, a DNA polymerase having strand displacement activity and at the same time lacking 5 '→ 3' exonuclease activity is used.

  Although the method for detecting the presence of the target DNA of the present invention has been described for each reaction step as described above, the reaction can be carried out in the presence of all the enzymes used in the present invention simultaneously. The reaction temperature is preferably in the range from about 30 degrees to about 70 degrees depending on the temperature conditions of the enzyme used. However, in order to suppress the production of non-specific amplification products and increase the reaction rate, the reaction temperature is as high as about 60 degrees. It is further desirable to be done.

  The present invention also includes the target DNA detection probe itself used in the method for detecting the presence of the target DNA. That is, in the target DNA detection probe of the present invention, the 3 ′ side portion is a base sequence complementary to the base sequence of one strand of the double-stranded target DNA, and the 5 ′ side is the base sequence of the target DNA. A target DNA detection probe that is an arbitrarily selectable base sequence (referred to as a 5 ′ arm sequence) that is not complementary, and the target DNA detection probe hybridizes with a restriction enzyme recognition sequence present in the target DNA. It has a base sequence portion capable of soybean, and the base sequence portion contains at least one base sequence modified so as to be protected from restriction enzyme cleavage.

The present invention is a kit in which the following materials (1) to (3) are combined. The kit includes a form in which all the materials (1) to (3) below are present in one system, and at least one of the materials (1) to (3) is independent of the remaining materials. Selected from existing forms.
(1) The 3 ′ side portion is a base sequence complementary to the base sequence of one strand of the double-stranded target DNA, and the 5 ′ side is an arbitrarily selectable base that is not complementary to the base sequence of the target DNA. A probe for detecting a target DNA which is a sequence (referred to as a 5 ′ arm sequence), and the probe for detecting a target DNA has a base sequence portion capable of hybridizing with a restriction enzyme recognition sequence present in the target DNA. A probe for detecting a target DNA comprising at least one base sequence modified so that the base sequence portion is protected from cleavage by a restriction enzyme;
(2) an enzyme comprising a restriction enzyme and a strand displacement DNA polymerase, and
(3) Deoxyribonucleotide triphosphate containing dATP, dGTP, dCTP and dTTP.

  The method for detecting the presence of target DNA using the target DNA detection probe in the present invention detects or quantifies an immunological ligand or immunological anti-ligand, specifically, an antigen or an antibody. Applicable to the method. For example, (1) a solid phased immunological ligand, a sample suspected of containing an immunological anti-ligand having a binding property to the solid phased immunological ligand, An immune complex is formed by contacting an immunological ligand having binding ability with an immunological anti-ligand and binding a target DNA of an arbitrary base sequence, and (2) By detecting the target DNA by applying the target DNA detection probe by the method of the present invention to the target DNA in the complex, an immunological anti-ligand is detected based on the detected target DNA. It can be quantified. In the immunological anti-ligand detection or quantification method, the reactivity of the immobilized immunological ligand and the immunological ligand bound to the target DNA may be different or the same. There may be.

  In the present invention, “immunological ligand” and “immunological anti-ligand” have binding properties to each other and mean either an antigen or an antibody.

  In the immunological anti-ligand detection or quantification method, the immobilization means shown in Japanese Patent No. 3197277 is applied to the means for immobilizing either antibody or antigen on the solid phase. Also good. That is, an antibody or antigen bound with an oligonucleotide (the former) is prepared, while an oligonucleotide complementary to the oligonucleotide is immobilized on a solid phase (the latter), and the former oligonucleotide is prepared. Prepare the solid phase immobilized with the antigen or antibody by reacting the introduced antibody or antigen with the latter complementary oligonucleotide-immobilized antibody in advance, or perform the binding reaction by mixing the former and the latter In parallel with this, the immune reaction may be advanced to immobilize the antibody or antigen on the solid phase.

  The method for detecting or quantifying the immunological ligand or immunological anti-ligand of the present invention can be specifically performed as follows. . For example, as shown in the schematic diagram of the antigen detection process in FIG. 6, (1) a solid-phased antibody is prepared, and (2) it is suspected that an antigen having a binding property is contained in the solid-phased antibody. By contacting the sample with the immobilized antibody, the antigen is captured by the immobilized antibody. (3) Homogeneous or heterogeneous that binds at a site different from the binding site in the antigen bound to the immobilized antibody. An antibody, which binds to a target DNA having an arbitrary base sequence, is contacted with the antigen captured by the solid-phased antibody in the above step to form an immune complex, and (4) in the immune complex The target DNA is detected by applying the target DNA detection probe to the target DNA by the above method. Based on the detected target DNA, the antigen can be detected or quantified.

  In addition, the method for detecting or quantifying the immunological ligand or immunological anti-ligand of the present invention can be applied to antibody detection, for example, antibody tests such as viral infections. The method for detecting or quantifying the antibody of the present invention includes, for example, (1) preparing an immobilized antigen and (2) binding to the immobilized antigen, as shown in the schematic diagram of the antibody detection process of FIG. A sample suspected of containing an antibody having a sex by contacting the immobilized antigen to cause the antibody to be captured by the immobilized antigen; (3) an antibody having binding properties to the antibody, An antibody complex bound to a target DNA of an arbitrary base sequence is brought into contact with the antibody captured by the solid-phased antigen in the above step to form an immune complex, and (4) against the target DNA in the immune complex The target DNA is detected by applying the target DNA detection probe by the above method. Based on the detected target DNA, the antibody can be detected or quantified.

  Another method for detecting or quantifying the antibody of the present invention other than the above is, for example, as shown in the schematic diagram of the antibody detection process of FIG. Immunization by contacting a sample suspected of containing a binding antibody with an antigen that binds to the antibody and binds to a target DNA of an arbitrary base sequence. A complex is formed, and (2) the target DNA is detected by applying the target DNA detection probe to the target DNA in the immune complex by the method according to any one of claims 1 to 7. Based on the detected target DNA, the antibody can be detected or quantified.

1) Preparation of double-stranded target DNA FIG. 9 shows the base sequence of the verotoxin type 1 (VT1) gene fragment of Escherichia coli (O157 H7), which is the target DNA. In the base sequence of FIG. 9, a BsrS1 recognition sequence and a BsrS1 cleavage site are shown. The single-stranded base sequences constituting the double-stranded base sequence of the gene fragment shown in FIG. 9 are shown in SEQ ID NO: 1 and SEQ ID NO: 2. The nucleotide sequence of the VT1 gene was obtained from the NCBI database (accession number is LO4539). On the database, an oligonucleotide (Ssl) (SEQ ID NO: 1) having the same sequence as the 60-mer long nucleotide sequence located at +3781 to +3840 was synthesized. The base sequence includes a BsrS1 recognition sequence at a position of +3795 to +3801 on the database. Furthermore, an oligonucleotide (Ss2) (SEQ ID NO: 2) having a base sequence completely complementary to the base sequence was synthesized. Each synthesized oligonucleotide was dissolved in a TE solution (10 mM Tris-HCl, 1 mM EDTA pH 8.0) to a concentration of 100 pmol / μL. After 10 μL of each oligonucleotide solution was taken out and mixed, heat treatment was performed at 94 ° C. for 3 minutes, and the temperature was gradually returned to room temperature. By this operation, it was confirmed by ethidium bromide staining after electrophoresis using 2% agarose gel (manufactured by NuSieve GTG Agarose, Cambrex) that each oligonucleotide hybridized and formed a double-stranded DNA. The electrophoresis chart is shown in FIG. According to FIG. 10, when Ssl and Ss2 were hybridized, a stronger band was detected in the polymer region than the mobility when each was migrated alone. This result indicates that Ss1 and Ss2 were hybridized to form a double-stranded DNA, the electrophoretic mobility was changed, and staining with ethidium bromide was strengthened. It was confirmed that DNA was formed.

2) Preparation of target DNA detection probe The target DNA detection probe has an arbitrary base sequence (5 'arm sequence) from the 5' end to the downstream, and double-stranded target DNA downstream of the 5 'arm sequence. It was designed to have a base sequence necessary for hybridizing with one strand and to further contain a restriction enzyme recognition sequence on the 5 ′ side of the base sequence. When the target DNA detection probe hybridizes with one strand of the target DNA to form a double-stranded DNA, a restriction enzyme cleavage site is bound to protect the target DNA detection probe from the restriction enzyme cleavage. A phosphorothioate linkage was made. FIG. 11 and SEQ ID NO: 3 show the base sequence of the target DNA detection probe synthesized for detecting the target DNA. In particular, FIG. 11 shows each site of the 5 ′ arm sequence, the BsrS1 recognition sequence, and the base sequence complementary to the base sequence of the target DNA in the target DNA detection probe.

  The base sequence of the target DNA detection probe has a 5 ′ arm sequence (TGTATAGTCGT) shown in SEQ ID NO: 4 having 12 bases from the 5 ′ end to the downstream, and downstream of the 5 ′ arm sequence is a double-stranded target DNA. A probe for detecting a target DNA having a total length of 54 mer, in which the base sequence shown in SEQ ID NO: 5 necessary for hybridizing one strand (TACAACACTGGGAsTGATCTCAGTGGGCGTTTCTTATGTAATGAC) was designed. The 5 'side in the base sequence has the sequence shown in SEQ ID NO: 6 (ACTGGGAsT) recognized by the restriction enzyme BsrS1 when forming a double strand. Here, s represents a phosphorothioate bond, and sT represents T modified with a phosphorothioate bond. In order to make the recognition by BsrS1 more reliable, the structure up to the sixth base upstream of the BsrS1 recognition sequence is hybridized with the target DNA.

  Therefore, the 5 ′ arm sequence from the 5 ′ end of the target DNA detection probe to the 7 bases upstream of the BsrS1 recognition site is the 5 ′ arm sequence, and the target DNA extends from the 6th base upstream of the BsrS1 recognition site to the 3 ′ end of the target DNA detection probe. It becomes a base sequence for hybridizing with. In addition, a phosphorothioate bond (denoted by s) is introduced so that the bond between AT of the target DNA detection probe, which is a cleavage site by BsrS1, is protected from cleavage.

3) Detection of double-stranded target DNA In constructing the double-stranded target DNA detection system, the following reagents were prepared.

10 × buffer: 500 mM KCl, 1.0% Triton X-100, solution containing 100 mM Tris-HCl pH 9.0 10 mM dNTP
10 μM target double-stranded DNA
50 μM target DNA detection probe Enzyme solution: 1M NaCl, 50 mM MgCl ₂ , 4U Bst DNA polymerase Large Fragment (manufactured by New England Biolabs), 5U BsrS1 (manufactured by Promega)

  The above components were reacted in the following reaction system. That is, 5 μL of the 10 × buffer, 1 μL of the 10 mM dNTP, 1 μL of the 10 μM target double-stranded DNA, 1 μL of the 50 μM target DNA detection probe were added to 37 μL of sterile water, heat-treated at 94 ° C. for 3 minutes, Incubated for 1 minute at ° C. Subsequently, 5 μL of the enzyme solution was added, and the reaction was allowed to proceed by incubating at 60 ° C. for 90 minutes. Detection of the amplification product was confirmed by ethidium bromide staining after agarose gel electrophoresis (FIG. 12).

  If the theoretical reaction proceeds, the amplification product should be a complementary strand of the sequence upstream from the BsrS1 recognition sequence of the target DNA detection probe, and this amplification product should contain the complementary strand of the 5 'arm sequence. In order to confirm that the amplification product is a sequence containing a complementary strand of the 5 ′ arm sequence, an oligonucleotide having the same base sequence as the base sequence of up to 15 bases from the 5 ′ end to the downstream of the target DNA detection probe ( Dp) was synthesized. The oligonucleotide and the amplification product were gently returned to room temperature after heat treatment at 94 ° C., and it was confirmed that hybridization occurred by ethidium bromide staining after electrophoresis.

  That is, the oligonucleotide TGTATAGTCGGTTAC (Dp: abbreviation) shown in SEQ ID NO: 9 having a 5 ′ arm sequence of 50 μM dissolved in TE was prepared, and 10 μL of the sample after the reaction was prepared by 10 μL. Mix and hold at 94 ° C. for 3 minutes. After gently returning to room temperature, the band was detected by ethidium bromide staining after agarose gel electrophoresis. The electrophoresis chart is shown in FIG. As shown in FIG. 13, when a sample obtained by subjecting the reaction product and Dp to hybridization was migrated, a strong band with a different mobility was detected compared to the band detected when the reaction product alone was migrated. This indicates that the reaction product and Dp were hybridized, the mobility was changed, and at the same time a double strand was partially formed, so that ethidium bromide was easily bound and the band became dark. That is, when the reaction sample is hybridized with Dp, a strong band is detected in the low-molecular region compared to when the reaction sample is migrated alone, so the reaction product surely contains the complementary strand of the 5 ′ arm sequence. Can be confirmed.

  In FIG. 10 described in the section “1) Preparation of double-stranded target DNA” in the examples, double-stranded DNA obtained by hybridizing single-stranded DNAs having complementary base sequences to each other is obtained by electrophoresis. However, in FIG. 13, it can be seen that the double-stranded DNA has moved to the lower molecular side than the single-stranded DNA. This is because the mobility of the single-stranded DNA is related to the intramolecular higher order structure in addition to the molecular weight, so the mobility varies greatly depending on the base composition, and thus the double-stranded DNA that should be a polymer is 1 This is probably because it is located on the lower molecular side than the double-stranded DNA.

1) Preparation of DNA binding antibody i. F (ab ′) ² of rabbit antibody against hepatitis B virus Rabbit polyclonal antibody (anti-HBsAg antibody) (manufactured by Immunoprobe) against hepatitis B surface antigen was digested with pepsin as follows, and F (Ab ′) ² was prepared. That is, 2.7 mg of the antibody dissolved in 0.8 mL of 0.1 M sodium acetate buffer pH 4.5, 0.05 mg of 2% pepsin (manufactured by BOEHRINGER MANNEIM), 0.05 mL of 0.1 M What was melt | dissolved in the sodium acetate buffer pH4.5 was mixed, and it incubated at 37 degreeC for 12 hours. Subsequently, F (ab ′) ² is separated from the pepsin digest of Fc by gel filtration column chromatography using 0.1 M phosphate buffer pH 6.0 (the carrier is AcA44 manufactured by BIOSEPRA). And concentrated to 1.14 mg / mL by ultrafiltration using Amicon Ultra-4 10,000 MWCO (trade name, manufactured by MILLIPORE) to obtain a concentrated solution of F (ab ′) ² .

ii. Introduction of maleimide group into oligonucleotide 200 nmols of the oligonucleotide of SEQ ID NO: 7 having an amino group introduced at the 5 ′ end dissolved in 127 μL of TE, and 3.1 mg of 1N- (6-Maleimidocaproyloxy) succinimide What was dissolved in 25.4 μL of dimethylformamide (manufactured by Dojindo Laboratories) was mixed and incubated at 37 ° C. for 1 hour to introduce a maleimide group via the amino group of the oligonucleotide. Subsequently, 50 μL of 3M sodium acetate pH 5.2 and 1 mL of ethanol were added to this mixed solution, incubated at −80 ° C. for 30 minutes, and then centrifuged at 15000 rpm, 10 minutes at 4 ° C. to introduce a maleimide group oligonucleotide. And the supernatant was removed, and then dissolved in 100 μL of 0.1 M phosphate buffer pH 6.0 to obtain a maleimide group-introduced oligonucleotide solution.

iii. Preparation of Fab ′ by F (ab ′) ² Reduction Operation i. 0.1M in containing the prepared F (ab ') ² of 1.3mL concentrate (in the concentrated solution F (ab') ² is included 1.48 mg), the 100mM mercaptoethylamine (5 mM EDTA in 133μL Fab 'was prepared by reducing S—S bond to SH group by mixing for 90 minutes at 37 ° C. and dissolving in phosphate buffer (pH 6.0). The obtained Fab ′ was purified by gel filtration column chromatography (using a PD10 column manufactured by Pharmacia) using 0.1 M phosphate buffer pH 6.0 containing 5 mM EDTA, and Amicon Ultra-4 10,000 MWCO ( The solution was concentrated to 4.8 mg / mL by ultrafiltration using a trade name, manufactured by MILLIPORE.

iv. Binding of Fab ′ and Maleimide Group-Introduced Oligonucleotide 40 nmols of maleimide group-introduced oligonucleotide prepared in the above step ii. and Fab ′ 0.72 mg prepared in the above step iii. are mixed and incubated at 37 ° C. for 1 hour. Thus, a binding reaction between the maleimide group and the SH group was performed to prepare an Fab-linked oligonucleotide, that is, a DNA-bound anti-HBsAg antibody. The obtained DNA-binding anti-HBsAg antibody was purified by gel filtration column chromatography using 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA (the carrier used is AcA44 manufactured by BIOSEPRA), and then Amicon Ultra. The solution was concentrated to 0.37 mg / mL by ultrafiltration using 10,000 MW (trade name, manufactured by MILLIPORE).

2) Construction of immunoassay system for detecting oligonucleotide after immune reaction by sandwich method Surface antigen (HBsAg) of hepatitis B virus using microtiter plate (F8 MAXISORP LOOSE (trade name) manufactured by Nalge Nunc International) An immunoassay system was constructed as follows.

  That is, 100 μL of anti-HBsAg antibody dissolved in a phosphate buffer solution (PBS: abbreviation) of pH 7.4 at a concentration of 50 μg / mL is dispensed into the well of the polystyrene microtiter plate and incubated at 23 ° C. for 2 hours. Thus, an anti-HBsAg antibody was immobilized on the well surface. After discarding the solution in the well and washing with 400 μL of PBS, 200 μL of PBS containing 0.5% BSA was added to the well and incubated at 23 ° C. for 1 hour to block the well surface. The blocking solution was discarded, and 100 μL of hepatitis B virus surface antigen (HBsAg) prepared with PBS to a concentration of 100 μg / mL was added to the wells and incubated at 23 ° C. for 2 hours. Separately, as a negative control, PBS without HBsAg added and incubated at 23 ° C. for 2 hours was also prepared. After discarding the HBsAg-containing solution and washing the wells with 400 μL of PBS three times, 100 μL of DNA-bound anti-HBsAg antibody (oligonucleotide-introduced antibody solution) dissolved in PBS at a concentration of 50 μg / mL was added, and 2 at 23 ° C. Incubated for hours. Next, the oligonucleotide-introduced antibody solution was discarded, washed with 400 μL of PBS three times, and then PBS was discarded. A DNA-bound anti-HBsAg antibody DNA was used as a target DNA to carry out a nucleic acid signal amplification reaction as follows.

That is, 100 μL of nucleic acid signal amplification buffer (6 mM Tris-HCl pH 7.9, 6 mM MgCl ₂ , 150 mM NaCl, 100 μg / mL BSA) is shown in SEQ ID NO: 8 and the detailed arrangement is shown in FIG. After adding 1 μL of a target DNA detection probe (10 pmol / μL) and preincubating for 3 minutes at 60 ° C., 20 U of BsrS1 and 1.5 U of BstDNA Polymerase Large Fragment were added and incubated at 60 ° C. for 2 hours. The target DNA detection probe shown in FIG. 14 has a 5 ′ arm sequence of 18 bases from the 5 ′ end, and a recognition sequence by the restriction enzyme BsrS1 and a sequence complementary to the target DNA sequence are linked downstream thereof. In order to avoid cleavage by BsrS1, a phosphorothioate bond is introduced into the bond between AA in the BsrS1 recognition sequence.

  A photograph showing the result of ethidium bromide staining by performing 2% agarose gel electrophoresis using the sample after incubation is shown in FIG. According to FIG. 15, no band was observed in the reaction product (lane 1) of the immunoassay system to which no HBs antigen was added, whereas there was no band in the reaction product (lane 2) of the immunoassay system to which HBs antigen was added. Is recognized.

  Further, an experiment for confirming that the band shown in FIG. 15 contains a complementary strand of the 5 'arm sequence was performed as follows. That is, it is shown in SEQ ID NO: 9 having a sequence of 15 bases downstream from the 5 ′ end in the 5 ′ arm sequence of 50 μmols / μL (see FIG. 14) and the reaction product 3 μL of the immunoassay system to which HBs antigen has been added. Were mixed with 5 μL of oligonucleotide (Dp), incubated at 94 ° C. for 3 minutes, and then gently returned to room temperature. When this sample was stained with ethidium bromide after agarose gel electrophoresis, a strong band was observed as compared to when both were run alone. A photograph of the obtained ethidium bromide staining is shown in FIG. In FIG. 16, lane 1 is Dp250 pmols, lane 2 is an immunoassay reaction product 3 μL added with HBs antigen, and lane 3 is a sample obtained by hybridizing Dp250 pmols and HBs antigen reaction product 3 μL. .

  According to FIG. 16, almost no band is observed in Dp alone (lane 1) or reaction product alone (lane 2), but in the case where Dp and reaction product are hybridized (lane 3), double-stranded DNA is formed. It can be confirmed that the reaction product surely contains the complementary strand of the 5 ′ arm sequence. According to Example 2, it can be confirmed that the nucleic acid signal amplification reaction can be performed by using the DNA introduced into the antibody as the target DNA, and can be applied to the detection of an immune reaction.

  The method for detecting the presence of the target DNA of the present invention is useful in the research field, clinical field, and industrial field for detecting a trace amount of DNA or screening screening for a trace amount of DNA. Since the method for detecting the presence of the target DNA of the present invention can be applied to the detection of an antibody or an antigen, it is useful for an immunological test. Specifically, detection of the presence of viruses causing disease (eg, SARS virus) and bacteria, detection of the presence of food poisoning bacteria in foods, harmful bacteria such as Legionella in pools, public baths, bathrooms, etc. It can also be used to detect bacteria and viruses, and to inspect virus and bacterial weapons.

FIG. 3 is a process diagram for producing a single-stranded target DNA by dissociating a double-stranded target DNA having a restriction enzyme recognition sequence by denaturation treatment. FIG. 4 is a process diagram for hybridizing a single-stranded target DNA detection probe and a single-stranded target DNA by a temperature drop. FIG. 3 is a process diagram for performing an extension reaction on the 3 ′ side of a target DNA detection probe with a polymerase (strand displacement DNA polymerase) and cleaving (nicking) only the target DNA side with a restriction enzyme. The strand displacement type DNA polymerase recognizes the 3 ′ end of the target DNA generated by nicking as the replication origin, and strand displacement is performed using the sequence upstream of the restriction enzyme recognition sequence including the 5 ′ arm sequence of the target DNA detection probe as a template. FIG. 3 is a process diagram in which a type polymerase reaction proceeds. A restriction enzyme B recognition sequence that is different from the restriction enzyme recognition sequence in the base sequence of the target DNA and that is not complementary to the base sequence of the target DNA when a preferred restriction enzyme recognition sequence is not present in the base sequence of the target DNA FIG. 3 is a process diagram in which a strand displacement polymerase reaction is allowed to proceed in a target DNA detection probe. It is the schematic of the detection process of the antigen of this invention. It is the schematic of the detection process of the antibody of this invention. FIG. 3 is a schematic view of another antibody detection process of the present invention. It is a figure which shows each area | region of the base sequence of the verotoxin type 1 (VT1) gene fragment of Escherichia coli (O157 H7) used as a target, a BsrS1 recognition sequence, and a BsrS1 cleavage site. It is a photograph which shows ethidium bromide dyeing | staining after electrophoresis for confirming that each oligonucleotide hybridized and formed double stranded DNA. The figure shows the base sequence and 5 ′ arm sequence of the target DNA detection probe synthesized for detecting the target DNA, and in particular shows the BsrS1 recognition sequence and the base sequence complementary to the base sequence of the target DNA. is there. It is a photograph which shows what ethidium bromide dye | stained after electrophoresis of the amplification product. It is a photograph which shows the ethidium bromide dyeing | staining after electrophoresis which compares the thing which the reaction product and Dp hybridized, and the thing of a reaction product only. Shows the base sequence of the target DNA detection probe synthesized for detecting the DNA of the DNA-binding anti-HBsAg antibody as the target DNA, and in particular, the 5 'arm sequence, the BsrS1 recognition sequence, the base complementary to the target DNA base sequence It is a figure which shows each area | region of an arrangement | sequence. It is a photograph which shows what carried out the ethidium bromide dyeing | staining after electrophoresis of the reaction product of an immunoassay system. It is a photograph which shows the ethidium bromide dyeing | staining after the electrophoresis which compares the thing which hybridized the reaction product of the immunoassay system, and Dp, and the thing of only the reaction product.

Claims (13)

Using a target DNA detection probe in which a base sequence complementary to the base sequence of the target DNA and a non-complementary base sequence that can be arbitrarily selected are combined, and complementary to the base sequence of the target DNA in the target DNA detection probe A method for detecting the presence of a target DNA, comprising amplifying a nucleic acid signal comprising an oligonucleotide containing a base sequence complementary to an unfavorable base sequence. The target DNA detection probe has an arbitrarily selectable base sequence in which the 3 ′ side portion is a base sequence complementary to the base sequence of the target DNA and the 5 ′ side portion is not complementary to the base sequence of the target DNA The method for detecting the presence of a target DNA according to claim 1, wherein The target DNA detection probe has a base sequence portion capable of hybridizing with a restriction enzyme recognition sequence present in the target DNA, and the base sequence portion is modified so as to be protected from cleavage by a restriction enzyme. 2. The method for detecting the presence of a target DNA according to claim 1, comprising at least one sequence. (A) The 3 ′ side portion is a base sequence complementary to the base sequence of one strand of the double-stranded target DNA, and the 5 ′ side is an arbitrarily selectable base that is not complementary to the base sequence of the target DNA. A probe for detecting a target DNA which is a sequence (referred to as a 5 ′ arm sequence), and the probe for detecting a target DNA has a base sequence portion capable of hybridizing with a restriction enzyme recognition sequence present in the target DNA. And a probe for detecting a target DNA comprising at least one base sequence modified so that the base sequence portion is protected from restriction enzyme cleavage,
(B) hybridizing the target DNA detection probe and the target DNA;
(C) let the 3′-end extension reaction of the hybridized target DNA detection probe obtained in the step (b) be performed,
(D) Nicking is performed by allowing a restriction enzyme to act on the restriction enzyme recognition site of the hybridized target DNA obtained in the step (c),
(E) by performing an extension reaction of the 3 ′ end of the target DNA with a strand displacement DNA polymerase using the 3 ′ end of the cleavage site of the target DNA generated by the nicking obtained in the step (d) as a replication start point Creating a double strand consisting of a complementary strand to the target DNA detection probe containing the 5 ′ arm sequence,
(F) Cleavage of the target DNA by nicking by causing a restriction enzyme to act on the double-stranded restriction enzyme recognition site obtained in the step (e),
(G) By performing extension reaction of the 3 ′ end of the target DNA with a strand displacement type DNA polymerase using the 3 ′ end of the cleavage site of the target DNA generated by the nicking obtained in the step (f) as a replication origin. Producing a double-stranded DNA consisting of a complementary strand to a DNA detection probe containing a 5 'arm sequence, and simultaneously releasing an oligonucleotide containing a base sequence complementary to the base sequence of the 5' arm sequence;
(H) Presence of a target DNA characterized by amplifying a nucleic acid signal comprising an oligonucleotide containing a base sequence complementary to the base sequence of the 5 ′ arm sequence by repeating the steps (f) and (g) How to detect. 5. The method for detecting the presence of target DNA according to claim 4, wherein both the restriction enzyme and the strand displacement type DNA polymerase have heat resistance. The method for detecting the presence of a target DNA according to claim 3 or 4, wherein the restriction enzyme recognition sequence is a sequence recognized by a restriction enzyme selected from the group consisting of BsrSI, BsrI, BstOI, BstNI, BsmAI, BslI and BsoBI. . 6. The method for detecting the presence of a target DNA according to claim 4 or 5, wherein the strand displacement DNA polymerase is selected from Bca and Bst. The 3 ′ side portion is a base sequence complementary to the base sequence of one strand of the double-stranded target DNA, and the 5 ′ side is not complementary to the base sequence of the target DNA. A target DNA detection probe (referred to as an arm sequence), and the target DNA detection probe has a base sequence portion capable of hybridizing with a restriction enzyme recognition sequence present in the target DNA, and A probe for detecting a target DNA, wherein the base sequence part comprises at least one base sequence modified so as to be protected from restriction enzyme cleavage. A kit in which the following materials (1) to (3) are combined, wherein all the materials (1) to (3) are present and contained in one system, and (1) to (3) A kit selected from a form in which at least one or more materials are present independently of the rest of the materials:
(1) The 3 ′ side portion is a base sequence complementary to the base sequence of one strand of the double-stranded target DNA, and the 5 ′ side is an arbitrarily selectable base that is not complementary to the base sequence of the target DNA. A probe for detecting a target DNA which is a sequence (referred to as a 5 ′ arm sequence), and the probe for detecting a target DNA has a base sequence portion capable of hybridizing with a restriction enzyme recognition sequence present in the target DNA. A probe for detecting a target DNA comprising at least one base sequence modified so that the base sequence portion is protected from cleavage by a restriction enzyme;
(2) an enzyme comprising a restriction enzyme and a strand displacement DNA polymerase, and
(3) Deoxyribonucleotide triphosphate containing dATP, dGTP, dCTP and dTTP. (1) an immobilized immunological ligand;
A sample suspected of containing an immunological anti-ligand having binding properties to the immobilized immunological ligand;
An immunological ligand that binds to the immunological anti-ligand and binds a target DNA of an arbitrary base sequence;
To form an immune complex by contacting
(2) A method for detecting or quantifying an immunological anti-ligand based on the target DNA detected by the method according to any one of claims 1 to 7 with respect to the target DNA in the immune complex. (1) Prepare an immobilized antibody,
(2) By contacting a sample suspected of containing an antigen having a binding property to the immobilized antibody with the immobilized antibody, the antigen is captured by the immobilized antibody;
(3) Homologous or heterogeneous antibody that binds at a site different from the binding site in the antigen bound to the immobilized antibody, and binds the target DNA of any base sequence to the solid phase in the above step An immune complex is formed by contact with the antigen captured by the conjugated antibody,
(4) The target DNA detected by detecting the target DNA by applying the target DNA detection probe to the target DNA in the immune complex by the method according to any one of claims 1 to 7. A method for detecting or quantifying an antigen based on the above. (1) Prepare an immobilized antigen,
(2) By contacting a sample suspected of containing an antibody having binding property to the immobilized antigen with the immobilized antigen, the antibody is captured by the immobilized antigen;
(3) An immune complex is formed by contacting an antibody having binding ability to the target DNA having an arbitrary base sequence with the antibody captured by the immobilized antigen in the above step. And
(4) The target DNA detected by detecting the target DNA by applying the target DNA detection probe to the target DNA in the immune complex by the method according to any one of claims 1 to 7. A method for detecting or quantifying antibodies based on the above. (1) an immobilized antigen;
A sample suspected of containing an antibody capable of binding to the immobilized antigen;
An antigen having a binding property to the antibody and binding to a target DNA having an arbitrary base sequence;
To form an immune complex by contacting
(2) The target DNA detected by detecting the target DNA by applying the target DNA detection probe to the target DNA in the immune complex by the method according to any one of claims 1 to 7. A method for detecting or quantifying antibodies based on the above.