JP2006180755A - Method for labeling nucleic acid, primer set used for the method and labeled nucleic acid prepared by the method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for preparing a labeled nucleic acid with attaining high detection sensitivity without depending on the location of a probe sequence in detecting the nucleic acid by a hybridization method using a detection probe immobilized on a solid phase. <P>SOLUTION: Two or more nucleic acid species containing an oligonucleotide labeled at 5'terminal are simultaneously prepared by extending a nucleic acid chain at 3' terminal of each primer using a primer set comprising two or more labeled oligonucleotide species prepared based on two or more complementary partial base sequences to the base sequence of one chain nucleic acid used as a template nucleic acid. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a nucleic acid labeling method for preparing a labeled nucleic acid having a base sequence complementary to a partial region of the entire base sequence of a template nucleic acid by an extension reaction, and a labeled oligonucleotide primer used in the method A set, as well as labeled nucleic acids prepared by the method. In particular, from a template nucleic acid contained in a sample, a labeled nucleic acid having a base sequence complementary to a partial region of the entire base sequence of the template nucleic acid is prepared by extension reaction, and probe hybridization is performed. The present invention relates to a nucleic acid labeling method suitably used when preparing a labeled nucleic acid sample for nucleic acid detection by reaction.

  As represented by the Human Genome Project, various analyzes have been made on the base sequences of various organisms such as genomic genes and mitochondrial genes. Furthermore, research on the relationship between the elucidated genes and the mechanisms of life activities, various diseases, diseases, genetic constitutions, etc. is also progressing, and research results have been reported one after another. From these research results, knowing the presence or absence of a specific gene or the abundance (expression level) of its expression product enables more detailed characterization and typing of factors such as various diseases and diseases. It has been found that useful information can be obtained. In addition, more detailed characterization and typing of various diseases, diseases, and other factors will make it easier to select effective treatment methods and effectively use them not only for disease diagnosis but also for their treatment. It has also been verified that it is possible.

  As a method for detecting the presence or absence of a specific gene in a specimen, as well as its expression level, many methods have been proposed and used in practice. When the base sequence of a gene or nucleic acid molecule to be detected is known, the most widely used technique is to select a characteristic partial base sequence from the base sequence of the gene or nucleic acid molecule, This is a method for detecting the presence or content of a nucleic acid chain containing such a characteristic partial base sequence using a nucleic acid probe having the complementary base sequence. Specifically, a DNA strand for a probe having a complementary base sequence is prepared in advance, a gene or nucleic acid molecule contained in a sample is made into a single-stranded nucleic acid, and a hybridization reaction is performed with the DNA probe. This is a probe hybridization method in which the presence or absence and the amount of formation of a hybrid is detected by some method.

  In this method of detecting a specific nucleic acid molecule by the probe hybridization method, the hybridization reaction itself can be performed either in the liquid phase or on the solid surface as long as the formed hybrid can be separated. Good. For example, when a hybridization reaction is performed on a solid phase surface, a DNA probe is previously immobilized on the solid phase surface by binding or adsorption, and the formed hybrid is immobilized on the solid phase. To separate. At that time, a labeled nucleic acid that has been labeled with a certain detectable labeling substance on a nucleic acid sample contained in a specimen, formed a hybrid with a DNA probe, and fixed and separated on a solid phase The presence / absence of a chain and the amount thereof are measured using a signal resulting from the labeling substance. Further, as a solid phase (base material) for fixing a DNA probe, a chip using a flat substrate surface such as glass or metal or a bead using a microparticle surface is a body surface.

  In the probe hybridization method, the first reason why a hybridization reaction using a DNA probe immobilized on a solid phase is preferred is that B / F separation is easy. In addition, since a DNA probe immobilized at a predetermined position on the solid phase is used, the detection region can be physically miniaturized, and as a result of specifying the detection region, highly sensitive measurement is possible. Become. At this time, multiple items can be detected at the same time by physically isolating the fixing positions of a plurality of types of DNA probes. Furthermore, when a form of a DNA chip or a DNA microarray in which a predetermined amount of DNA probe is immobilized on a solid phase is selected in advance, there is an advantage that its handling and application become easier.

  For example, Affymetrix Inc. in the United States uses a nucleic acid labeled with a fluorescent dye to act on oligo DNA synthesized on a planar substrate, and a hybrid formed by the hybridization reaction is obtained by fluorescence derived from the fluorescent label. A detection method is proposed (see Patent Document 1). By using a DNA array composed of oligo DNA synthesized on this substrate and a fluorescence detection method using a fluorescent dye label, it is possible to detect the presence and amount of a specific nucleic acid contained in a specimen.

  Fuji Film also applied a technique for immobilizing multiple types of separately prepared DNA probes using amino groups pre-introduced on the surface of the substrate, and produced a DNA array. Thus, a labeled 22-mer single-stranded DNA is detected (see Patent Document 2).

  By the way, in a method of detecting a specific nucleic acid by using a DNA probe immobilized on a solid phase as described above and applying a hybridization reaction to form a hybrid and detecting a specific nucleic acid, a nucleic acid as a sample is labeled in advance. It is necessary to attach it. As a method for incorporating a labeling substance into a sample nucleic acid, for example, there is a method of adding a labeled deoxynucleotide (eg, Cy3-dUTP) when preparing a PCR amplification product.

  In addition, when preparing a PCR amplification product, ATCG4 types of deoxynucleotides (dATP, dCTP, dGTP, dTTP: generically dNTP) and labeled deoxynucleotides (for example, Cy3-dUTP), which are substrates for the DNA polymerase enzyme, are used. There is also a method in which labeled deoxynucleotides are incorporated into the produced amplification product by preparing and adjusting the final concentration of each dNTP. Sake Brewery Co., Ltd. substitutes a fluorescently labeled nucleotide for one of the four substrate nucleotides of the DNA polymerase enzyme, and incorporates the labeling substance into the amplified product (see Patent Document 3).

In addition, a method of labeling a nucleic acid by performing a PCR reaction using a pre-labeled primer is known (see Patent Document 4). When this pre-labeled primer is used, there is an advantage that the amount ratio of the labeling substance to be applied can be controlled per molecule of the labeled nucleic acid to be prepared. Is preferred.
US Pat. No. 6,410,229 JP 2001-128683 A Japanese Patent No. 3001919 Japanese Patent No. 2649793

  A nucleic acid detection method using a hybridization reaction using a DNA probe immobilized on a solid phase has advantages such as higher sensitivity than other nucleic acid detection methods. On the other hand, when compared with a detection method that utilizes a hybridization reaction in a liquid phase, there remains a problem that is difficult to control accompanying the reaction on the solid phase.

  For example, unlike a cDNA array having a base length equal to that of a nucleic acid to be detected and having a complementary DNA full length immobilized as probe DNA, the probe DNA is compared with the base length of the nucleic acid to be detected. In a system with a short base length, a hybrid of a probe DNA having a short base length fixed on a solid phase and a nucleic acid to be detected is formed, and then B / F separation is performed, and the hybrid is attached to the nucleic acid to be detected. When the fluorescence intensity of a fluorescent label is measured, there is a problem that the fluorescence intensity is greatly influenced by the position of the binding site with the probe DNA on the nucleic acid to be detected.

  Specifically, when a fluorescent label is attached to the 5 ′ end of the nucleic acid to be detected, the measured fluorescence intensity tends to decrease as the binding site with the probe DNA moves away from the 5 ′ end. Therefore, when the binding site with the probe DNA is selected near the 3 ′ end of the nucleic acid to be detected and when the binding site with the probe DNA is selected near the 5 ′ end, the 5 ′ end of the nucleic acid to be detected is selected. It has been found that there is a significant difference in the fluorescence intensity measured from the fluorescent labels attached to the. Although the detailed mechanism of this phenomenon has not been elucidated at this stage, a fluorescent label is attached to the 5 ′ end, which is prepared by a PCR reaction using a pre-labeled primer to label a nucleic acid. When using a nucleic acid, it is a factor that impairs its high quantitativeness. Empirically, when performing detection using a DNA probe immobilized on a solid phase for a nucleic acid having a fluorescent label at the 5 ′ end, the binding site with the DNA probe is determined at the 5 ′ end. It has been confirmed that the influence of the above phenomenon can be avoided by setting in the vicinity.

  However, in order to detect the detection target nucleic acid with higher selectivity, it is essential to select the base sequence of the DNA probe in a complementary manner to the base sequence portion specific to the detection target nucleic acid. In many cases, the base sequence portion specific to the nucleic acid to be detected does not exist in the vicinity of the 5 'end of the base sequence of the nucleic acid to be detected. In that case, the measured fluorescence intensity is reduced, but a specific nucleotide sequence located at a position far from the 5 ′ end is used, or the selectivity is lowered, but the measured fluorescence intensity is Whether to use a base sequence portion having difficulty in specificity in the vicinity of the 5 ′ end, which is high, or to select one of the choices of dual exclusion, it becomes a problem. That is, when a nucleic acid with a fluorescent label at the 5 ′ end, which is prepared by a method of labeling a nucleic acid by performing a PCR reaction using a pre-labeled primer having excellent quantitative properties, a solid phase In many cases, high detection sensitivity due to the use of the DNA probe immobilized thereon and high selectivity due to the specificity of the probe base sequence are not compatible.

  For example, regarding a specific patient suffering from an infectious disease, consider the case of identifying which causative bacterium among a plurality of types of infectious bacterium causing an infectious disease. When identifying multiple types of pathogenic bacteria based on genetic information, for example, among the rRNAs commonly held by each bacteria, among the 16s rRNA base sequences, the partial base sequences characteristic of individual bacteria are It can be used for identification. Specifically, the 16s rRNA gene contained in the collected genome of each pathogenic bacterium is used as a template, and PCR amplification is performed using PCR primers having a base sequence common to these pathogenic bacteria. A double-stranded DNA sample corresponding to the 16s rRNA gene is prepared. On the other hand, the base sequences of the 16s rRNA gene-derived double-stranded DNA fragments prepared by the PCR amplification are compared with each other, and a unique base sequence portion is selected for each strain. One or more DNA probes are prepared. A double-stranded DNA sample corresponding to the 16s rRNA gene prepared using a common PCR primer is subjected to a hybridization reaction with a DNA probe for identifying individual strains to detect the presence or absence of hybrids and the amount thereof. To do. When a hybrid is formed and the amount exceeds a desired level, a double-stranded DNA whose partial base sequence complementary to the base sequence of the DNA probe for specifying the strain corresponds to a 16s rRNA gene It is confirmed to be present on either strand of DNA.

  In general, when comparing bacteria that are closely related in taxonomy, the homology in the base sequence of the 16s rRNA gene is high, and the base sequence portion unique to each strain exists only in a very limited position. Not a few. As a result, a unique nucleotide sequence portion used for identification of a strain of a causative fungus is used for detection in double-stranded DNA corresponding to a 16s rRNA gene prepared using a common PCR primer. The position may be far from the 5 ′ end of the single-stranded DNA. At that time, when a single-stranded DNA having a fluorescent label at the 5 ′ end is used for detection, when a hybrid is formed with a detection DNA probe immobilized on a solid phase, fluorescence resulting from the fluorescent label is generated. There are situations where the intensity is only “weak” and cannot be measured. That is, it may be a factor that greatly restricts the presence or absence of hybrid formation and the quantitativeness of the amount.

  Usually, using a nucleic acid contained in a specimen as a template, a DNA fragment corresponding to a gene to be detected is prepared as a PCR amplification product and used as a primary sample for actual probe hybridization. This primary sample substantially contains only a double-stranded DNA fragment corresponding to the gene to be detected, prepared by PCR amplification. Next, the double-stranded DNA fragment is dissociated into two single-stranded DNA fragments that are complementary to each other, and then a DNA strand extension reaction is performed using a pre-labeled primer to label the nucleic acid. By applying this technique, a nucleic acid (in particular, a single-stranded DNA fragment) having a fluorescent label at the 5 ′ end capable of forming a hybrid with a detection DNA probe is prepared.

  On the other hand, the binding site (position) of the detection DNA probe is selected based on the base sequence of the double-stranded DNA fragment corresponding to the gene to be detected contained in the primary sample. In this case, even if the position far from the 5 ′ end of the DNA fragment corresponding to the gene to be detected is selected as the binding site (position) of the detection DNA probe, the above method is applied. A hybrid formed by a hybridization reaction between a nucleic acid (particularly a single-stranded DNA fragment) having a fluorescent label at the 5 ′ end and a detection DNA probe immobilized on a solid phase. In the body, the fluorescence caused by the fluorescent label is expected to develop a means for preparing a nucleic acid (especially a single-stranded DNA fragment) having a fluorescent label at the 5 ′ end, which can exhibit sufficient fluorescence intensity. It is rare.

  The present invention solves the above-mentioned problems, and an object of the present invention is to detect the gene to be detected that is present in a primary sample prepared in advance by PCR amplification as a binding site (position) of a DNA probe for detection. Even when a position far from the 5 ′ end of the corresponding DNA fragment is selected, a fluorescent label is produced at the 5 ′ end, which is prepared by applying a nucleic acid chain extension reaction using the labeled primer. In the hybrid formed by the hybridization reaction between the attached nucleic acid (especially a single-stranded DNA fragment) and the detection DNA probe immobilized on the solid phase, the fluorescence resulting from the fluorescent label is sufficient It is an object of the present invention to provide a method for preparing a nucleic acid (particularly, a single-stranded DNA fragment) having a fluorescent label at the 5 ′ end, which can exhibit high fluorescence intensity.

  In addition, an object of the present invention is to provide a labeled primer set that is preferably used in the method for preparing a nucleic acid (particularly, a single-stranded DNA fragment) having a fluorescent label at the 5 ′ end. Is also included.

  In order to solve the above-mentioned problems, the present inventors have intensively studied, studied and studied.

  Since the amount of nucleic acid contained in a sample, for example, a genomic gene collected from inside a cell is very small, a PCR amplification reaction is usually performed using the nucleic acid (genomic gene) contained in the sample as a template. Thus, a DNA fragment corresponding to the gene to be detected is prepared in advance. In nucleic acid detection using an actual probe hybridization method, a nucleic acid solution containing a double-stranded DNA fragment corresponding to the gene to be detected, amplified at a desired amplification rate by this PCR reaction, is used as a primary sample. Use.

  In the hybrid formed by the probe hybridization reaction, a single-stranded DNA fragment that binds to a DNA probe to form a hybrid is used to control the amount ratio of the labeling substance to be added per molecule of the hybrid. Using a labeled oligonucleotide having a fluorescent label introduced at the 5 ′ end in advance as a primer, extending the nucleic acid strand at the 3 ′ end, and a labeled nucleic acid (single-stranded DNA fragment) It is necessary to. In this nucleic acid chain extension reaction, the double-stranded DNA fragment corresponding to the gene to be detected is dissociated, and the corresponding complementary single-stranded DNA fragment (complementary strand) is used as a template for the 3 ′ end of the labeled oligonucleotide. A nucleic acid chain having the desired base sequence is generated. The labeled nucleic acid to be produced (single-stranded DNA) has a DNA fragment (oligonucleotide) of the labeled oligonucleotide itself used as a primer at its 5 'end. Therefore, when the nucleic acid strand extension reaction is performed using the same single-stranded DNA fragment (complementary strand) as a template, the nucleotide sequence of the labeled oligonucleotide itself (oligonucleotide) used as a primer is different. Naturally, the base sequence at the 5 ′ end of the labeled nucleic acid (single-stranded DNA fragment) also differs. In particular, labeled oligos are used for complementary single-stranded DNA fragments (complementary strands) that serve as templates and are obtained by dissociating double-stranded DNA fragments corresponding to the gene to be detected contained in the primary sample. The DNA fragment (oligonucleotide) of the nucleotide itself shall have a complementary base sequence.

  Furthermore, as a labeled oligonucleotide to be used as a primer, at least two or more different base sequences that bind at different sites (positions) to a complementary single-stranded DNA fragment (complementary strand) as a template Two or more kinds of labeled nucleic acids (single-stranded DNA fragments) obtained by using a DNA fragment (oligonucleotide) as a reference, based on the 5 ′ end of the DNA fragment corresponding to the gene to be detected ), The present inventors have conceived that the positions of the 5 ′ ends can be different from each other. In addition, as described above, two or more kinds of labeled nucleic acids (prepared so that the positions of the 5 ′ ends differ from each other with respect to the 5 ′ end of the DNA fragment corresponding to the gene to be detected ( When a mixture of single-stranded DNA fragments) is used, a position far from the 5 ′ end of the DNA fragment corresponding to the gene to be detected is selected as the binding site (position) of the DNA probe for detection. Even so, one of a plurality of labeled nucleic acids (single-stranded DNA fragments) contained in the mixture has a detection DNA probe binding site (position) in the vicinity of its 5 ′ end. I found what I can do. In addition to the above knowledge, the present inventors also have one of a plurality of nucleic acids (single-stranded DNA fragments) having a fluorescent label at the 5 ′ end and a detection DNA probe immobilized on a solid phase. The present invention was completed by verifying that the fluorescence resulting from the fluorescent label in the hybrid formed by the hybridization reaction with the above showed sufficient fluorescence intensity.

That is, the nucleic acid labeling method according to the present invention includes:
A means for preparing a labeled nucleic acid for nucleic acid detection using a probe hybridization method,
Using a single-stranded nucleic acid prepared from a nucleic acid contained in a sample as a template nucleic acid,
Using a labeled oligonucleotide having a base sequence complementary to the partial base sequence contained in the base sequence of the template nucleic acid as a primer,
When a nucleic acid strand is extended to the 3 ′ end of the labeled oligonucleotide and an extension reaction is performed to prepare a nucleic acid inherent in the labeled oligonucleotide at the 5 ′ end to form a labeled nucleic acid. ,
As a primer for the extension reaction, using a primer set comprising at least two or more kinds of labeled oligonucleotides that differ in the complementary base sequences constituting the oligonucleotide,
With respect to a single-stranded template nucleic acid used for extension of a nucleic acid strand at the 3 ′ end of one labeled oligonucleotide contained in the primer set,
At least one of the other labeled oligonucleotides contained in the primer set has a base sequence complementary to the partial base sequence contained in the base sequence of the single-stranded template nucleic acid. A method for labeling nucleic acid, comprising:

  In the present invention, when detecting nucleic acid using a detection DNA probe immobilized on a solid phase, for example, a DNA microarray, the detection target gene is assumed to be a binding site (position) of the detection DNA probe. Even when a position far from the 5 ′ end of the corresponding DNA fragment is selected, the nucleic acid labeled with a fluorescent label at the 5 ′ end prepared by using the nucleic acid labeling method of the present invention (one In a hybrid formed by a hybridization reaction between one of a plurality of types of single-stranded DNA and a detection DNA probe immobilized on a solid phase, the fluorescence resulting from the fluorescent label exhibits sufficient fluorescence intensity Therefore, there is an advantage that detection sensitivity and accuracy can be improved.

In the present invention, the invention of the nucleic acid labeling method described above, that is,
A means for preparing a labeled nucleic acid to be used for nucleic acid detection using a probe hybridization method,
Using a single-stranded nucleic acid prepared from a nucleic acid contained in a sample as a template nucleic acid,
Using a labeled oligonucleotide having a base sequence complementary to the partial base sequence contained in the base sequence of the template nucleic acid as a primer,
When preparing a nucleic acid containing the labeled oligonucleotide at the 5 ′ end by extending the nucleic acid strand at the 3 ′ end of the labeled oligonucleotide to prepare a nucleic acid containing the labeled oligonucleotide at the 5 ′ end. ,
As a primer for the extension reaction, a primer set comprising at least two kinds of labeled oligonucleotides that are different in the complementary base sequence constituting the oligonucleotide is used,
With respect to a single-stranded template nucleic acid used for extending a nucleic acid strand at the 3 ′ end of one labeled oligonucleotide contained in the primer set,
At least one of the other labeled oligonucleotides contained in the primer set has a base sequence complementary to the partial base sequence contained in the base sequence of the single-stranded template nucleic acid. As an example of a preferred embodiment in the nucleic acid labeling method characterized by comprising the above oligonucleotides, the following embodiment can be mentioned.

Specifically, in the nucleic acid labeling method according to the present invention,
As the extension reaction, an asymmetric PCR reaction can be used. Alternatively, a PCR reaction can be used as the extension reaction.

On the other hand, the labeled oligonucleotide is
The oligonucleotide is preferably labeled using one or more labels selected from the group consisting of fluorescent substances and biotin.

In that case, in the nucleic acid labeling method according to the present invention,
Nucleic acid detection using the probe hybridization method,
It is desirable to use a hybridization reaction for a DNA probe fixed or adsorbed on the solid surface. In particular, the DNA probe immobilized or adsorbed on the solid surface is
More preferably, it is in the form of a DNA chip or a DNA microarray.

As the template nucleic acid,
It is desirable to select a form using a single-stranded nucleic acid prepared as a PCR product from a nucleic acid contained in a specimen.

The nucleic acid labeling method according to the present invention includes:
The nucleic acid to be detected is more preferably used in the form of a 16s rRNA gene derived from bacteria.

In addition, the present invention also provides an invention of a labeled nucleic acid that can be produced using the nucleic acid labeling method according to the present invention described above,
That is, the labeled nucleic acid according to the present invention is
It is a labeled nucleic acid prepared by the nucleic acid labeling method of the present invention that selects any one of the above-described configurations.

Furthermore, the present invention also provides an invention of a primer set used in the above-described nucleic acid labeling method according to the present invention,
That is, the primer set according to the present invention is:
It is a primer set comprising at least two or more kinds of labeled oligonucleotides that can be used in the nucleic acid labeling method according to the present invention for selecting any one of the above-described configurations.

For example, the primer set according to the present invention is:
Based on the base sequence of the template nucleic acid,
A probe-corresponding site on the base sequence of the template nucleic acid, in which the base sequence of the probe DNA used for nucleic acid detection using the probe hybridization method is selected;
About at least two kinds of sites complementary to the primer on the base sequence of the template nucleic acid having a base sequence complementary to the base sequence of the at least two or more kinds of labeled oligonucleotides,
When defining the relative position of at least two kinds of complementary sites to the primer based on the number of bases up to the presence position of at least two kinds of complementary positions to the primer, using the probe corresponding site as a reference point,
The primer set is characterized in that the base sequences of the at least two or more kinds of labeled oligonucleotides are selected so that the relative positions of at least two kinds of sites complementary to the primer are different from each other. preferable.

As a specific example,
As the at least two or more kinds of labeled oligonucleotides contained in the primer set,
Sequence 1 to Sequence 9 below:
Sequence 1; 5 'actgctgcctcccgtaggagtctgg 3'
Sequence 2; 5 'cgtattaccgcggctgctggcacg 3'
Sequence 3; 5 'gcgtggactaccagggtatctaatcctgtttg 3'
Sequence 4; 5 'tcgaattaaaccacatgctccaccgcttgtgcggg 3'
Sequence 5; 5 'ggtaaggttcttcgcgttgc 3'
Sequence 6; 5 'ttgacgtcatccccaccttcctcc 3'
Sequence 7; 5 'ccattgtagcacgtgtgtagccc 3'
Sequence 8; 5 'tggtgtgacgggcggtgtgtacaag 3'
Sequence 9; 5 'taccttgttacgacttcacccca 3'
A primer set comprising a labeled oligonucleotide having at least two or more base sequences selected from the group consisting of can be shown as a preferred example.

  Hereinafter, the present invention will be described in more detail.

  First, a labeled nucleic acid to which a nucleic acid labeling method according to the present invention is applied is a single-stranded nucleic acid that can form a hybrid with a DNA probe for detection when detecting a nucleic acid using a probe hybridization method. A nucleic acid molecule having a portion is labeled with a label used for detection.

  The single-stranded nucleic acid portion capable of forming a hybrid with the detection DNA probe is contained in the base sequence of the template nucleic acid using a single-stranded nucleic acid prepared from the nucleic acid contained in the sample as a template nucleic acid. Using a labeled oligonucleotide having a base sequence complementary to the partial base sequence as a primer, it is produced by extending a nucleic acid chain to the 3 ′ end of the labeled oligonucleotide. In this nucleic acid chain extension reaction, the labeled oligonucleotide used as a primer becomes the 5 ′ end of the finally produced single-stranded nucleic acid moiety, so that the label contained in the labeled oligonucleotide is labeled. The nucleic acid is added to the 5 ′ end of the nucleic acid.

  In the present invention, for example, a bacterial nucleic acid molecule can be used as the nucleic acid contained in the specimen. In other words, for example, a sample containing a bacterium that is the source of such a bacterium-derived nucleic acid molecule is subjected to a process of extracting a bacterium-derived nucleic acid molecule to be detected, and used as a specimen. Examples of samples containing bacteria that are the source of nucleic acid molecules derived from such bacteria include blood derived from animals such as humans and livestock, body fluids such as sputum, gastric juice, vaginal secretions, oral mucus, urine, and feces. Samples derived from living organisms such as effluents, or food poisoning, contaminated foods, environmental waters such as drinking water and hot spring water, etc. It is done. In addition, tissue samples collected from animals and plants that have been confirmed to be infected by bacteria in quarantine at the time of import / export are also included in the subject. Usually, a sample containing a bacterium that is a source of such a bacterium-derived nucleic acid molecule contains the bacterium in a live or dead sterilized state, and therefore, an extraction process for a bacterium-derived nucleic acid molecule present in the cell. After removing unnecessary solid components, the nucleic acid molecules are separated and collected in a soluble fraction to obtain a specimen containing nucleic acid.

  At that time, the nucleic acid molecule contained in the sample may be either an RNA molecule or a DNA molecule. When the target nucleic acid molecule is an RNA molecule, a DNA molecule complementary to the base sequence, that is, a cDNA molecule, is prepared using the RNA molecule as a template and a reverse transcriptase reaction. For example, once the total RNA is separated from the nucleic acid molecules contained in the sample, a corresponding cDNA library is constructed. Using the cDNA derived from the RNA molecule to be detected contained in the constructed cDNA library as a template, a DNA fragment containing the desired base sequence region (target region) was prepared by PCR amplification reaction. It is preferably used as a “single-stranded nucleic acid prepared from a nucleic acid contained in

  On the other hand, when the target nucleic acid molecule is a DNA molecule, for example, a DNA fragment containing a desired base sequence region (target region) is prepared using enzyme digestion with a restriction enzyme. It can also be used as a “single-stranded nucleic acid prepared from”. For example, since only a very small amount of DNA genome is present per cell, a DNA fragment containing a desired nucleotide sequence region (target region) is prepared in advance by PCR amplification using the DNA genome as a template. , “Single-stranded nucleic acid prepared from nucleic acid contained in specimen” is preferably used.

  When using, as a primary sample, a nucleic acid solution containing a double-stranded DNA fragment corresponding to a gene to be detected, amplified at a desired amplification rate by the PCR reaction based on nucleic acid molecules contained in a sample. The double-stranded DNA fragment corresponding to the gene to be detected is subjected to heat treatment, and the dissociated single-stranded DNA fragment is used as “single-stranded nucleic acid prepared from nucleic acid contained in the specimen”. The base sequence of this single-stranded DNA fragment has its 5 'end and 3' end determined by the base sequence of the PCR primer used in the PCR reaction, and its sequence length is also determined. A single-stranded DNA fragment whose total base sequence is known is used as a template nucleic acid, and a labeled oligonucleotide having a base sequence complementary to the partial base sequence contained in the base sequence of the template nucleic acid is used. It is produced by extending a nucleic acid strand to the 3 ′ end of the labeled oligonucleotide, using it as a primer.

  In the nucleic acid labeling method according to the present invention, at least two kinds of labeled oligonucleotides are used as primers for the extension reaction. Both of these two types of labeled oligonucleotides are capable of extending a nucleic acid chain at the 3 'end of the oligonucleotide using the single-stranded DNA fragment whose total base sequence is known as a template nucleic acid. The oligonucleotide and the nucleic acid chain extended to the 3 ′ end thereof can be either an RNA chain or a DNA chain, but usually a DNA chain to which an extension reaction using a DNA polymerase can be applied. Is preferably used.

  At least two or more of the labeled oligonucleotides have base sequences complementary to the partial base sequence contained in the base sequence of the single-stranded nucleic acid used as the template nucleic acid. It is said. In addition, at least two or more of the labeled oligonucleotides are selected so that the nucleotide sequences of the oligonucleotides are different from each other. The primer set according to the present invention is a primer mixture comprising at least two or more kinds of labeled oligonucleotides having different nucleotide sequences from each other. The content ratio of the plurality of kinds of labeled oligonucleotides contained in the primer set is a plurality of kinds of labeled nucleic acids prepared by extending a nucleic acid chain using the labeled oligonucleotides as primers. Preferably, the yields of are selected to be substantially equal.

  That is, in principle, depending on the selection of the base sequence of the labeled oligonucleotide, the hybridization efficiency with the single-stranded nucleic acid used as the template nucleic acid and the extension of the nucleic acid chain to its 3 ′ end are completed. Differences occur in the reaction time required. Usually, a nucleic acid chain extension reaction using a primer with a single-stranded nucleic acid as a template nucleic acid is performed in an annealing step for hybridizing the primer to the single-stranded nucleic acid, and the nucleic acid chain is transferred to the 3 ′ end of the primer. It comprises a nucleic acid strand synthesis step that performs extension, and a denaturing step that separates the single-stranded nucleic acid containing a primer at the 5 ′ end and the single-stranded nucleic acid of the template. The melting temperature (Tm) of a hybrid of a labeled oligonucleotide used as a primer and a single-stranded nucleic acid that is a template nucleic acid is significantly higher than the temperature of the nucleic acid strand synthesis step, The base sequence of the labeled oligonucleotide is selected so as to be significantly lower than the temperature of the denaturing step. At that time, in the plurality of types of labeled oligonucleotides included in the primer set, the melting temperature Tm is in the range of 45 ° C. to 80 ° C., and the difference between the melting temperatures Tm is 10 It is desirable to select the base sequence of each labeled oligonucleotide so that it is within ° C, preferably within 5 ° C. In general, the base length of the base sequence of each labeled oligonucleotide is desirably selected within the range of at least 15 bases to 40 bases. Even if the base length of the base sequence of the labeled oligonucleotide is selected to be less than 15 bases, for example, 13 bases, the melting temperature Tm can be set within the aforementioned temperature range depending on the type of base sequence. On the other hand, even when the base length of the base sequence of the labeled oligonucleotide exceeds 40 bases, for example, 45 bases, the melting temperature Tm may be set within the above temperature range depending on the type of base sequence. It is possible. However, the base length of the base sequence exceeds 40 bases and becomes long, and not only the complementary base sequence portion present in the target single-stranded nucleic acid, but also this complementary base sequence portion. This increases the probability of misfit hybridization to other single-stranded nucleic acids having a base sequence similar to. That is, in addition to the target single-stranded nucleic acid, using other single-stranded nucleic acid as a template nucleic acid, there is a probability that a nucleic acid chain extension reaction will occur at the 3 ′ end of the labeled oligonucleotide that has caused the misfit hybridization. And will not be suitable for the purposes of the present invention.

  In the plurality of types of labeled oligonucleotides included in the primer set, it is desirable to appropriately select the content ratio of the plurality of types of labeled oligonucleotides in consideration of the melting temperature Tm. When the difference between the melting temperatures Tm is within the above preferable range, the content ratio of the plurality of types of labeled oligonucleotides is that the melting temperature Tm is the highest and the Tm is the lowest. It is desirable that the content ratio be appropriately set so that the content ratio is in the range of 1: 1 to 1: 0.1.

  When labeling is performed using a reaction for producing a PCR product using a forward primer and a reverse primer based on a template nucleic acid, the above-mentioned multiple types of labels can be selected by selecting within the above range. For any of the attached oligonucleotides, a plurality of suitably labeled nucleic acids are prepared. The positional relationship between the plurality of types of labeled oligonucleotides and the probe setting position varies depending on the base sequence of the template nucleic acid, but the nucleic acid chain extends beyond the probe setting position by a normal PCR reaction. Is preferably a distance (base length) that can be performed without any problem, and at least the distance separating the two is preferably in the range of 10,000 base lengths or less.

  In the plurality of types of labeled oligonucleotides contained in the primer set, the labeling substances to be applied to the individual labeled oligonucleotides are attached so as to be a constant amount per oligonucleotide molecule. For example, a form in which one molecule of the labeling substance is attached to one oligonucleotide molecule is generally a more preferable form.

  Moreover, it is desirable to introduce the labeling substance to be attached to each labeled oligonucleotide to a site that does not inhibit hybridization with the single-stranded nucleic acid used for the template nucleic acid by the oligonucleotide. At the same time, after hybridization with a single-stranded nucleic acid used as a template nucleic acid, when performing a nucleic acid chain elongation reaction to the 3 ′ end of the oligonucleotide, binding of a nucleic acid chain synthase such as a DNA polymerase used, It is desirable to introduce it at a site that does not inhibit the nucleic acid chain elongation reaction. Accordingly, the labeling substance to be attached to each labeled oligonucleotide generally has a more preferable form in which it is introduced into the 5 'end of the oligonucleotide. Specifically, it is more preferable to use a labeling substance that can be introduced in an equivalent manner by synthesizing the oligonucleotide in advance and then covalently binding it to the 5 'end.

  Examples of labeling substances that can be introduced equivalently by covalently binding to the 5 'end include various fluorescent substances used as fluorescent labels, biotin and digoxigenin. Biotin reacts with various biotin-binding labeling enzyme proteins and enables detection using the enzyme activity of the labeling enzyme protein as an index. Digoxigenin can also be detected by reacting various digoxigenin-bound labeled enzyme proteins and using the enzyme activity of the labeled enzyme protein as an index. Various fluorescent materials used as fluorescent labels enable highly sensitive detection by using an optical detection means for observing the fluorescence intensity derived from such fluorescent materials. In addition, the detection method using the enzyme activity of the labeled enzyme protein as an index can also use optical detection means by using various color development reactions, and highly sensitive detection is possible. And

  In particular, when the form of a DNA chip or a DNA microarray is used as a DNA probe immobilized on a solid phase, the DNA probe is present at a high density per unit area. Therefore, a hybrid of a labeled nucleic acid and a DNA probe is also required to be detected with good reproducibility using a label in a state where it exists at a high density per unit area. In that case, the detection using the enzyme activity of the labeled enzyme protein as an index requires caution so as not to cause variations in the supply of the substrate substance used for the enzyme reaction. On the other hand, when various fluorescent materials used as fluorescent labels are used, the uniformity of the excitation light intensity can be achieved much more easily than the uniform supply of the substrate material. From this viewpoint, when a form of a DNA chip or a DNA microarray is used, the use of a fluorescent label is a more suitable form. Any conventionally known fluorescent dye can be used as the fluorescent dye that can be used for the fluorescent label. Among these, from the viewpoint of quantum yield, light resistance, gas resistance, chemical stability, and characteristics of DNA polymerase as a substrate, FITC, FAM, Cy3, Cy5, Cy5.5, Cy7, TAMRA, Dabcyl , ROX, TET, Rhodamine, Texas Red, HEX, Cyber Green, and the like can be suitably used in the present invention.

  Although it is possible to label the plurality of types of labeled oligonucleotides contained in the primer set with different types of labeled substances for each type of labeled oligonucleotide, it is easy to detect. In view of this, it is preferable to label using the same type of labeling substance. That is, using the plurality of types of labeled oligonucleotides, a plurality of types of labeled nucleic acids are prepared using the same single-stranded nucleic acid as a template nucleic acid. In probe hybridization, a specific base sequence is used. There is a possibility that one or more of a plurality of kinds of labeled nucleic acids form a hybrid with respect to a DNA probe having For example, when two kinds of labeled nucleic acids form a hybrid with respect to a DNA probe having a specific base sequence, if the labeling substances used in the two kinds of labeled nucleic acids are different, It is necessary to detect individual labeling substances and then add the results. On the other hand, if the labeling substances used in the two kinds of labeled nucleic acids are the same, the detection of the labeling substances and the sum of the results are performed at a time, so that the detection operation becomes simple.

  In the nucleic acid labeling method according to the present invention, by using the plurality of types of labeled oligonucleotides, a nucleic acid chain extension reaction to the 3 ′ end of each labeled oligonucleotide is performed using the same single-stranded nucleic acid as a template nucleic acid. As a result, a mixture containing a plurality of labeled nucleic acids is produced. In this nucleic acid chain extension reaction, a considerable amount of the plurality of types of labeled oligonucleotides contained in the primer set is consumed, but at the end of the reaction, it was not consumed in the reaction solution. There is a substantial residual amount of species labeled oligonucleotides. Upon detection of a target labeled nucleic acid, a plurality of types of labeled oligonucleotides remaining in this mixture form a hybrid with a DNA probe immobilized on a DNA chip or DNA microarray. It becomes a factor that hinders detection accuracy and quantitativeness. Therefore, it is preferable to purify the target labeled nucleic acid by removing a plurality of types of labeled oligonucleotides remaining in the mixture before the probe hybridization reaction.

  On the other hand, the detection DNA probe can form a hybrid with the target labeled nucleic acid, but the labeled oligonucleotide is substantially free from hybridization with the labeled oligonucleotide. It is more preferable to select the base sequence of the probe and the base sequence of the oligonucleotide part of the labeled oligonucleotide. For the purpose of the present invention, as the base sequence of the detection DNA probe, a partial base sequence specific to the nucleic acid to be detected is selected in advance from the base sequence of a single-stranded nucleic acid used as a template nucleic acid. . At least one of a plurality of types of labeled nucleic acids prepared by the nucleic acid labeling method according to the present invention forms a hybrid with the detection DNA probe. In this case, in principle, if the site where the detection DNA probe hybridizes is located in the nucleic acid strand that is extended to the 3 ′ end of the labeled oligonucleotide used as a primer in the labeled nucleic acid, the labeled DNA is labeled. Since formation of a hybrid between the oligonucleotide part of the oligonucleotide and the DNA probe for detection is avoided, this is a preferred form. That is, at least one of the plural types of labeled oligonucleotides included in the primer set used in the nucleic acid labeling method according to the present invention is preliminarily selected from the base sequence of a single-stranded nucleic acid used as a template nucleic acid. It is preferable to have a base sequence complementary to the partial base sequence located on the 3 ′ end side with respect to the selected partial base sequence for the DNA probe.

  In particular, at least one of the plurality of types of labeled oligonucleotides is located on the 3 ′ end side of a base sequence of a single-stranded nucleic acid used as a template nucleic acid with reference to the partial base sequence for the DNA probe. It is more preferable to select the base sequence of the labeled oligonucleotide so that the interval (base length) to the partial base sequence is within 3000 base lengths, more preferably within 1000 base lengths. In other words, for a labeled nucleic acid prepared by extending a nucleic acid strand at the 3 ′ end of the labeled oligonucleotide, the distance (base) from the 5 ′ end to the position where the DNA probe hybridizes. Length) is within 1000 bases. For example, the binding site of a DNA probe immobilized on a solid phase is near the 5 'end of a nucleic acid labeled with a fluorescent label at the 5' end. Is achieved, and empirically, the fluorescence resulting from the fluorescent label at the 5 ′ end is measured at a high intensity.

  When detecting a nucleic acid to be detected using a plurality of types of detection DNA probes, a partial base sequence for the plurality of types of DNA probes is selected from the base sequence of a single-stranded nucleic acid used as a template nucleic acid. Pre-selected. At that time, it is difficult to form a hybrid with any of the plural types of labeled oligonucleotides included in the primer set with respect to the partial base sequence for each DNA probe. With respect to the partial base sequence selected within the range of 300 base lengths on the 3 ′ end side of the base sequence, at least one labeled oligonucleotide having a complementary base sequence is present. More preferably, the base sequence of the labeled oligonucleotide is selected.

  Specifically, in the base sequence of a single-stranded nucleic acid used as a template nucleic acid, when the partial base sequences for the respective DNA probes are arranged from the 3 ′ end side, the first sequence located closest to the 3 ′ end side The first labeled oligonucleotide having a complementary base sequence to the partial base sequence selected from the 3 'end side and within a range of 300 bases or less of the partial base sequence for the DNA probe Is selected. Next, after the partial base sequence for the first DNA probe, the partial base sequence for the second DNA probe located on the 5 ′ end side is further within the range of the 3 ′ end side and within 300 base lengths. At the same time, the base of the second labeled oligonucleotide having a base sequence complementary to the partial base sequence selected on the 5 ′ end side from the partial base sequence for the first DNA probe Select an array. Thereafter, a base of a labeled oligonucleotide having a base sequence complementary to the partial base sequence selected in a region sandwiched between the partial base sequences for two DNA probes located adjacent to each other Select the sequence sequentially. As a result, when detecting a nucleic acid to be detected using a plurality of types of detection DNA probes, the same number of labeled oligonucleotide base sequences as the number of types of the plurality of types of DNA probes can be selected. it can. A plurality of types of labeled nucleic acids prepared by the nucleic acid labeling method according to the present invention is a primer set comprising a plurality of types of labeled oligonucleotides whose base sequences are selected according to the selection procedure described above. It is more preferable to produce using

  Even when using a primer set comprising a plurality of types of labeled oligonucleotides whose base sequences are selected according to the selection procedure described above for the plurality of types of DNA probes, they are also included in the sample. Each labeled nucleic acid is prepared by extending a nucleic acid strand at the 3 ′ end of the labeled oligonucleotide using a single-stranded nucleic acid prepared from the nucleic acid as a template nucleic acid. At that time, the extension of the nucleic acid strand to the 3 ′ end of the labeled oligonucleotide is carried out at least on the 5 ′ end side of the hybridization site of the labeled oligonucleotide on the single-stranded nucleic acid used as the template nucleic acid. It is necessary to exceed the partial base sequence for the DNA probe that is located. That is, although it is necessary to exceed the partial base sequence for the DNA probe located on the 5 ′ end side of the hybridization site of the labeled oligonucleotide, it is not always necessary for the single-stranded nucleic acid used as the template nucleic acid. There is no need to extend the nucleic acid strand until the 5 'end is reached.

  Therefore, when using a primer set comprising a plurality of types of labeled oligonucleotides whose base sequences are selected according to the selection procedure described above, in the nucleic acid labeling method according to the present invention, to the 3 ′ end of the primer The reaction conditions in the extension reaction step for extending the nucleic acid strand are longer than the reaction time required for extending the nucleic acid strand until reaching the 5 ′ end of the single-stranded nucleic acid used as the template nucleic acid. It is possible to select significantly shorter. For example, the number of bases of a nucleic acid chain that can be extended in the nucleic acid chain synthesis step is selected in the range of 300 to 3000 bases, and in some cases, the reaction conditions are in the range of 300 to 1000 bases. Is possible.

  In addition, when using a primer set comprising a plurality of types of labeled oligonucleotides whose base sequences are selected according to the selection procedure described above, for a single-stranded nucleic acid used as a template nucleic acid, It is also possible to adopt a form in which plural kinds of labeled oligonucleotides are simultaneously hybridized on the single-stranded nucleic acid in the annealing step for hybridizing the primers. At that time, the extension of the nucleic acid chain to the 3 ′ end of each labeled oligonucleotide is such that another labeled oligonucleotide is hybridized to the 5 ′ end side of the single-stranded nucleic acid used as the template nucleic acid. Therefore, in principle, it becomes a form that cannot proceed beyond the site where other labeled oligonucleotides are hybridized. Even in that case, the extension of the nucleic acid strand to the 3 ′ end of the labeled oligonucleotide is performed at least on the 5 ′ end side of the hybridization site of the labeled oligonucleotide on the single-stranded nucleic acid used as the template nucleic acid. It satisfies the requirement of exceeding the partial base sequence for the DNA probe located in the above, and there is no problem at all.

  Furthermore, when the situation in which all of the plural types of labeled oligonucleotides contained in the primer set hybridize on the single-stranded nucleic acid used as the template nucleic acid is achieved, 3 ′ of each labeled oligonucleotide is obtained. The extension of the nucleic acid strand to the end is at least a partial base sequence for the DNA probe located on the 5 ′ end side of the hybridization site of the labeled oligonucleotide on the single-stranded nucleic acid used as the template nucleic acid However, it becomes a form which stops immediately before the next labeled oligonucleotide which is hybridized immediately after that. Each of the plurality of types of labeled nucleic acids prepared in this form contains a labeled oligonucleotide at the 5 ′ end, and the nucleic acid strand extended to the 3 ′ end is only the DNA probe type. It becomes a shape exhibiting high specificity capable of forming a hybrid. At the same time, such a labeled nucleic acid was immobilized on a solid phase with respect to a nucleic acid labeled with a fluorescent label at the 5 ′ end, for example, when selectively hybridized with only one DNA probe. The binding site of the DNA probe is achieved in the vicinity of its 5 ′ end, and empirically, the fluorescence resulting from the fluorescent label at the 5 ′ end is also measured at high intensity. That is, in view of the object of the present invention, it is one of the most preferable modes.

In the nucleic acid labeling method according to the present invention,
Using a single-stranded nucleic acid prepared from a nucleic acid contained in a sample as a template nucleic acid,
Using a labeled oligonucleotide having a base sequence complementary to the partial base sequence contained in the base sequence of the template nucleic acid as a primer,
When a nucleic acid strand is extended to the 3 ′ end of the labeled oligonucleotide and an extension reaction is performed to prepare a nucleic acid inherent in the labeled oligonucleotide at the 5 ′ end to form a labeled nucleic acid. ,
In the extension reaction of the nucleic acid strand to the 3 ′ end of the labeled oligonucleotide, it is not always necessary to extend the nucleic acid strand until the 5 ′ end of the single-stranded nucleic acid used as the template nucleic acid is reached. The extension reaction can be performed in the form of an asymmetric PCR reaction.

  Furthermore, on the single-stranded nucleic acid used as the template nucleic acid described above, the situation in which all of the plural types of labeled oligonucleotides contained in the primer set hybridize is used for the 3 ′ end. A forward primer corresponding to a partial base sequence existing just before the site where the next labeled oligonucleotide hybridizes on a single-stranded nucleic acid used as a template nucleic acid instead of a form in which the extension of the nucleic acid chain is stopped It is also possible to carry out a PCR reaction using the labeled oligonucleotide as a reverse primer.

  In the nucleic acid labeling method according to the present invention, as described above, when a nucleic acid to be detected is detected using a plurality of types of detection DNA probes, the nucleic acid labeling method includes a single-stranded nucleic acid used as a template nucleic acid. From this, nucleic acid detection using the probe hybridization method is carried out by immobilizing or adsorbing the partial base sequences for the plurality of types of DNA probes. It is particularly suitable for a form utilizing a hybridization reaction with a DNA probe. In particular, when the DNA probe fixed or adsorbed on the solid surface is in the form of a DNA chip or a DNA microarray, the advantages of the nucleic acid labeling method according to the present invention become more remarkable.

  Hereinafter, the present invention will be described in more detail with reference to more specific examples and preferred embodiments thereof.

  Specifically, an example in which the present invention is applied to the identification of a plurality of pathogenic bacteria that cause infections will be described.

  For the purpose of identifying the type of bacteria, it is possible to use a specific rRNA gene derived from each bacterium, particularly a 16s rRNA gene, as a target for nucleic acid detection using the probe hybridization method. In particular, regarding multiple types of infectious bacteria causing infections, the base sequence of 16s rRNA derived from the bacteria has been elucidated, and by comparing the base sequences of 16s rRNA derived from each of the pathogenic bacteria, Thus, a base sequence unique to each pathogenic bacterium can be selected and used as a probe.

  Identifying the type of causative bacteria present in a sample collected from a specific patient suffering from an infectious disease is an indispensable process when proceeding with treatment for the patient. However, in this nucleic acid detection process, since the type of the pathogenic bacteria present in the sample collected from the patient is not known, first, the genomic gene is extracted and separated from the bacteria present in the sample. And an original sample containing the genomic gene. In many cases, the amount of bacterial genomic genes contained in the original sample is such that it can be used for nucleic acid detection using the probe hybridization method, which requires detection accuracy of a certain level or more. Is an amount that does not reach much.

  Even in reality, there are many cases where there are a plurality of candidates for the causative bacteria that cause infection. Among these multiple types of pathogenic bacteria candidates, when identifying the pathogenic bacteria present in a sample collected from a patient, individual 16s rRNA genes derived from the multiple types of pathogenic bacteria candidates are identified. It is necessary to make a positive / negative determination using a probe hybridization method for multiple types of detection probes that have selected a base sequence unique to the pathogenic fungus. Therefore, a DNA fragment containing a part or the entire length of the 16s rRNA gene derived from bacteria is prepared as a PCR product by using a PCR amplification reaction using the bacterial genomic gene contained in the original sample as a template. At that time, since the type of the existing bacteria is unknown, a highly common region (conserve sequence) found in the 16s rRNA gene is subjected to PCR in a plurality of types of pathogenic bacteria candidates regardless of the species of the bacteria. It is necessary to use the universal PCR method as a primer.

  A highly common region that can be used as a PCR primer when preparing a DNA fragment containing a part or the entire length of the 16s rRNA gene as a PCR product using a universal PCR method for multiple types of pathogenic bacteria candidates Examples of (conserve sequence) include the following. That is, in the base sequence of 16s rRNA derived from Escherichia coli, in the 5 ′ → 3 ′ direction, 9-27, 105-123, 339-357, 515-531, 686-704, 785-805, 907-926, The base sequences located at 1099-1114, 1224-11241, 1391-1406, 1495-1510, 1525-1541 are highly common regions (conservation sequences) among multiple types of pathogenic bacteria. The design of an actual universal PCR primer includes the base sequences of these highly common regions (conserve sequences), and if necessary, refer to the base sequences within about 10 bases before and after the base sequence. It is desirable to design a mix primer capable of amplifying at a certain level or higher for any of the species of pathogenic bacteria.

  Although depending on the base length of the designed primer, the corresponding base sequence portion of the 16s rRNA gene used as a template is 1 with respect to the whole base sequence of the primer including the base sequence of the highly common region (conserve sequence). Even a sequence containing a mismatch in the range of -10 bases often functions as a PCR primer. Therefore, it is possible to use one type of primer, but in consideration of mismatch variation, by using a mixed primer containing multiple types of primers, it is more than a certain level in the overall multiple types of pathogenic bacteria candidates. It is preferable to maintain the amplification efficiency. Furthermore, even if it is other than the base sequence of the highly common region (conservation sequence), it is possible to cover a total of 50 types of target genes with a variation of up to 50 variations in the mix primer. If so, it is also possible to use a primer corresponding to a region where sequence commonality is slightly inferior. However, if the total number contained in the mix primer exceeds 50, the total amount of primer added to the reaction solution will be excessive, or if the total amount of primer is limited, the amount of individual primers will decrease, resulting in relative amplification. It becomes a factor causing a decrease in efficiency. In addition, if the total number contained in the mix primer exceeds 50, the ratio used for actual amplification will be low with respect to the total amount of primer added to the reaction solution, which is preferable from the viewpoint of cost and performance. is not.

  In the present invention, a single-stranded nucleic acid is used as a template nucleic acid, and a nucleic acid chain is extended to the 3 ′ end of a plurality of types of labeled oligonucleotides used as primers. When specifying the type of the causative bacteria present in the sample, a DNA fragment containing a part or the entire length of the 16s rRNA gene previously amplified to a certain level is used as a template nucleic acid using the universal PCR. It is preferable to use it. In particular, when an oligonucleotide having a label introduced at the 5 ′ end is used as a plurality of types of labeled oligonucleotides, either asymmetric PCR or normal PCR is used for the nucleic acid chain extension reaction to the 3 ′ end. It is also possible to use.

  On the other hand, the plurality of types of labeled oligonucleotides used at that time are also nucleic acid strands using a part or the entire 16s rRNA gene amplified by universal PCR as a template nucleic acid for any of a plurality of types of pathogenic bacteria candidates. It is desirable to select a primer sequence that includes the base sequence of the above-mentioned highly common region (consequence sequence) that can be extended. That is, a primer oligo designed based on the base sequence of a highly common region (conserve sequence) located on the 3 ′ end side (downstream) of a highly common primer sequence used in universal PCR Those in which a label is introduced at the 5 ′ end of a plurality of nucleotides are preferably used.

  Since the detection probe is selected so as to hybridize to the region sandwiched between the highly common regions (conserve sequence), the primer oligonucleotides of multiple types of nucleotide sequences themselves and the detection probe are: There is no concern of direct hybridization. On the other hand, in any one of the plurality of primer oligonucleotides, the detection probe can be hybridized to the 3 ′ end side (downstream) in the vicinity of the base sequence of the oligonucleotide. Concomitantly, a detection probe immobilized on a solid phase is designed to hybridize to a nucleic acid having a fluorescent label introduced at the 5 ′ end at a position far from the 5 ′ end. The problem of low fluorescence intensity observed from the hybrid that is found in the process is avoided.

For example, in a plurality of types of pathogenic fungi candidates, as an example of a plurality of types of labeled oligonucleotides designed to correspond to the base sequence of a highly common region (conserve sequence) in 16s rRNA gene,
Sequence 1 to Sequence 9 below:
Sequence 1; 5 'actgctgcctcccgtaggagtctgg 3'
Sequence 2; 5 'cgtattaccgcggctgctggcacg 3'
Sequence 3; 5 'gcgtggactaccagggtatctaatcctgtttg 3'
Sequence 4; 5 'tcgaattaaaccacatgctccaccgcttgtgcggg 3'
Sequence 5; 5 'ggtaaggttcttcgcgttgc 3'
Sequence 6; 5 'ttgacgtcatccccaccttcctcc 3'
Sequence 7; 5 'ccattgtagcacgtgtgtagccc 3'
Sequence 8; 5 'tggtgtgacgggcggtgtgtacaag 3'
Sequence 9; 5 'taccttgttacgacttcacccca 3'
Can be mentioned. That is, as the at least two or more kinds of labeled oligonucleotides contained in the primer set, a labeled oligonucleotide having at least two or more base sequences selected from the group consisting of the sequences 1 to 9 It is desirable to utilize a primer set comprising.

  When a single-stranded nucleic acid is used as a template nucleic acid and a labeled oligonucleotide hybridized to the template nucleic acid is used as a primer, and a nucleic acid chain extension reaction to the 3 ′ end thereof is performed, the nucleic acid chain to the 3 ′ end of the primer The extension reaction depends on an elementary process in which a nucleic acid chain synthesizing enzyme (for example, DNA polymerase) takes in NTPs as a substrate. As a result, the chain length of the nucleic acid chain extended during a certain reaction time shows a Poisson distribution. That is, a certain chain length shows a maximum probability and contains a considerable amount of products with a shorter nucleic acid chain length. For example, when the distance (base length) from the hybridization site of the primer to the 5 ′ end of the template nucleic acid is long, most of the nucleic acid chain that is extended to the 3 ′ end of the primer during a certain short reaction time is the template nucleic acid. However, there is very little that reaches the 5 ′ end. In that case, when the detection probe is selected at the 5 ′ end side of the primer hybridizing site in the base sequence of the single-stranded nucleic acid used as the template nucleic acid, Almost all products subjected to nucleic acid chain extension can be hybridized with the detection probe. On the other hand, when the detection probe is selected in the vicinity of its 5 ′ end in the base sequence of the single-stranded nucleic acid used as the template nucleic acid, the product obtained by extending the nucleic acid chain to the 3 ′ end of the primer Very few of these can form a hybrid with the detection probe. That is, the distance (base length) from the hybridization site of the primer to the 5 ′ end of the template nucleic acid is significant compared to the average strand length of the nucleic acid strand extended to the 3 ′ end of the primer during a certain short reaction time. However, the content ratio of products capable of forming a hybrid varies greatly depending on the position of the detection probe selected.

  On the other hand, by using multiple types of labeled oligonucleotides, the single strand nucleic acid used as the template nucleic acid is compared with the average strand length of the nucleic acid strand that is extended to the 3 ′ end of the primer during a certain short reaction time. Even when the nucleic acid length is significantly long, most of the nucleic acid strands that extend to the 3 ′ end of the labeled oligonucleotide that hybridize to a site not far from the 5 ′ end of the single-stranded nucleic acid are 5 'The end is reachable. Therefore, in the multiple types of labeled nucleic acids obtained by using multiple types of labeled oligonucleotides, there is a significant difference in the content ratio of products capable of forming a hybrid regardless of the selected position of the detection probe. There can be no state.

  Regardless of the selected position of the detection probe, it has an effect of averaging the content ratio of products capable of forming a hybrid, and in any of a plurality of labeled oligonucleotides, for detection at a site close to the 3 ′ end side Combined with the effect of hybridization of the probe, high detection sensitivity can be achieved without being influenced by the selection position of the detection probe.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the examples described below are examples of the best mode according to the present invention, but the technical scope of the present invention is not limited to the modes shown in these examples.

Example 1
<Preparation of probe DNA>
As a probe for selective detection of nucleic acid molecules derived from Pseudomonas aeruginosa, a probe DNA having the base sequence shown in Table 1 was designed.

  Specifically, based on the base sequence of the genomic region coding for 16s rRNA of Pseudomonas aeruginosa bacteria, the base sequences of 8 types of probes shown in Table 1 were selected. The base sequences of these probes are selected so as to be very specific to the bacteria and to expect sufficient hybridization sensitivity. At the same time, it is designed so that it can be expected that there is no variation in sensitivity when the hybridization sensitivity is compared between the base sequences of the probes.

  For each probe shown in Table 1, after synthesis of the DNA strand, a thiol group was introduced at the 5 'end of the nucleic acid according to a standard method as a functional group for immobilization on the DNA microarray. After introduction of the functional group, it was purified and lyophilized. The lyophilized probe for internal standard was stored in a freezer at −30 ° C.

  A probe DNA having the base sequence shown in Table 2 was designed as a probe for selective detection of nucleic acid molecules derived from Haemophilus influenzae by a similar design technique.

<Preparation of PCR Primer for amplification of nucleic acid chain in specimen>
A DNA primer having the nucleic acid sequence shown in Table 3 was designed as a PCR primer for amplifying 16s rRNA gene (target gene) derived from the pneumoniae used for detection of the pneumoniae.

  Specifically, a probe set that specifically amplifies a genomic portion encoding 16s rRNA derived from a Pseudomonas aeruginosa standard strain (ATCC 10145), that is, bases at both ends of a 16s rRNA coding region having a length of about 1500 bases Based on the sequence, a plurality of kinds of primers having a base sequence complementary thereto and having substantially the same melting temperature when hybridized with the portion were designed. The designed multiple types of primers have partial base sequence differences from each other. By using this mixed primer, it is possible to simultaneously amplify mutant strains and multiple 16s rRNA coding regions on the genome. Designed to.

  Each Primer shown in Table 3 is purified by high performance liquid chromatography (HPLC) after synthesis of the DNA strand. Three kinds of Forward Primers and three kinds of Reverse Primers were mixed and dissolved in TE buffer so that each Primer concentration would be a final concentration of 10 pmol / μl.

<Extraction of Pseudomonas aeruginosa DNA (model specimen)>
[Pretreatment of microorganism culture and genomic DNA extraction]
First, a Pseudomonas aeruginosa standard strain (ATCC 10145) was cultured according to a standard method.

1.0 ml (OD ₆₀₀ = 0.7) of this microorganism culture solution was collected in a 1.5 ml capacity microtube, and the cells were separated by centrifugation (8500 rpm, 5 min, 4 ° C.). After discarding the supernatant, 300 μl of Enzyme Buffer (50 mM Tris-HCl: pH 8.0, 25 mM EDTA) was added, and the cells were resuspended using a mixer. The bacterial cells were separated again from the resuspended bacterial cell solution by centrifugation (8500 rpm, 5 min, 4 ° C.). After discarding the supernatant, 50 μl of each of the following two enzyme solutions was added to the collected cells and resuspended using a mixer.

Lysozyme 50 μl for lysis treatment (enzyme concentration: 20 mg / ml in Enzyme Buffer)
N-Acetylmuramidase SG 50 μl (enzyme concentration: 0.2 mg / ml in Enzyme Buffer)

Next, the bacterial cell solution resuspended in the buffer solution containing the enzyme was allowed to stand in an incubator at 37 ° C. for 30 minutes, and the cell membrane of the microorganism was dissolved.

(Genome extraction / purification)
After lysis treatment of the cell membrane, extraction and purification of Genome DNA derived from the microorganism was performed according to the following procedure using a nucleic acid purification kit (MagExtractor-Genome-: manufactured by TOYOBO).

(Step 1)
First, 750 μl of the lysis / adsorption solution of the kit and 40 μl of magnetic bead solution are added to the microorganism suspension subjected to the cell membrane dissolution treatment, and vigorously stirred for 10 minutes using a tube mixer. By this operation, the contained double-stranded DNA molecule is adsorbed on the surface of the magnetic particle (bead).

(Step 2)
Next, the microtube is set on a separation stand (Magnetic Trapper) and allowed to stand for 30 seconds to collect magnetic particles on the wall surface of the tube. Thereafter, the supernatant is discarded while being set on the stand.

(Step 3)
Add 900 μl of the washing solution into the microtube and stir it for about 5 seconds with a mixer to resuspend it.

(Step 4)
Again, the microtube is set on a separation stand (Magnetic Trapper) and allowed to stand for 30 seconds to collect magnetic particles on the wall of the tube. Thereafter, the supernatant is discarded while being set on the stand.

(Step 5)
The washing operations in steps 3 and 4 are repeated to perform washing twice.

(Step 6)
After washing with the washing solution, 900 μl of 70% ethanol is added to the microtube, and the mixture is stirred for about 5 seconds with a mixer and re-suspended.

(Step 7)
Next, the microtube is set on a separation stand (Magnetic Trapper) and allowed to stand for 30 seconds to collect magnetic particles on the wall surface of the tube. Thereafter, the supernatant is discarded while being set on the stand.

(Step 8)
By repeating the washing operations in steps 6 and 7, the second washing is performed with 70% ethanol in total.

  After washing twice with 70% ethanol, 100 μl of pure water is added into the microtube, and the liquid containing the collected magnetic particles is stirred for 10 minutes with a tube mixer. By this operation, the double-stranded DNA molecules adsorbed on the surface of the magnetic particles (beads) are re-eluted in pure water.

Next, the microtube is set on a separation stand (Magnetic Trapper), and left to stand for 30 seconds to collect magnetic particles on the tube wall surface. Finally, the supernatant containing the double-stranded DNA molecules is collected in a new tube while being set on the stand.

(Inspection of recovered Genome DNA)
The solution containing the purified nucleic acid molecule derived from the collected microorganism (Pseudomonas aeruginosa ATCC 10145 strain) is subjected to agarose electrophoresis and absorbance measurement at 260/280 nm according to a conventional method, and Genome DNA contained in the solution Quality (contamination amount of low-molecular nucleic acid, degree of degradation) and recovery amount were tested.

  In this example, about 10 μg of Genome DNA was recovered, and Genome DNA degradation and rRNA contamination were not observed.

<Production of DNA microarray>
[1] Cleaning of glass substrate A synthetic quartz glass substrate (size: 25 mm (width) x 75 mm (length) x 1 mm (thickness), manufactured by Iiyama Special Glass Co., Ltd.) is placed in a heat-resistant and alkali-resistant rack, It was immersed in a cleaning solution for ultrasonic cleaning adjusted to a concentration. After soaking in the cleaning solution overnight, ultrasonic cleaning was performed for 20 minutes. Subsequently, the substrate was taken out, rinsed lightly with pure water (rinse cleaning), and then ultrasonically cleaned in ultrapure water for 20 minutes. Next, the substrate was immersed in a 1N aqueous sodium hydroxide solution heated to 80 ° C. for 10 minutes. Thereafter, the substrate was taken out and rinsed with pure water and subjected to ultrasonic cleaning in ultrapure water. A quartz glass substrate for a DNA chip that has been washed was prepared by the above washing operation.

[2] Surface treatment A silane coupling agent KBM-603 (manufactured by Shin-Etsu Silicone) was added to pure water to a concentration of 1%, and stirred at room temperature for 2 hours to dissolve uniformly. Subsequently, the washed quartz glass substrate was immersed in an aqueous silane coupling agent solution and left at room temperature for 20 minutes. The quartz glass substrate was pulled up and the surface was rinsed lightly with pure water, and then dried by blowing nitrogen gas on both sides of the substrate. Next, the dried substrate was baked in an oven heated to 120 ° C. for 1 hour to complete the binding treatment of the aminosilane coupling agent to the substrate surface. An amino group derived from the aminosilane coupling agent was introduced on the surface of the substrate.

  N-maleimidocaproyloxy succinimide (N- (6-Maleimidocaproyloxy) succinimido; hereinafter abbreviated as EMCS) manufactured by Dojindo Laboratories Ltd. in a 1: 1 mixed solvent of dimethyl sulfoxide and ethanol has a final concentration of 0. An EMCS solution dissolved to 3 mg / ml was prepared. After completion of the baking treatment, the quartz glass substrate was allowed to cool to room temperature. Next, the quartz glass substrate having amino groups introduced on the surface was immersed in the EMCS solution at room temperature for 2 hours. During this immersion treatment, the amino group introduced to the substrate surface by the aminosilane coupling agent treatment and the succinimide group of EMCS reacted, and the maleimide group derived from EMCS was introduced to the quartz glass substrate surface. The quartz glass substrate pulled up from the EMCS solution was washed with a 1: 1 mixed solvent of dimethyl sulfoxide and ethanol to remove unreacted EMCS. Further, after washing with ethanol, the surface-treated quartz glass substrate was dried in a nitrogen gas atmosphere.

[3] Probe DNA
The probe DNA for detection prepared in <Preparation of probe DNA> was dissolved in pure water, dispensed into micro vials so that the final concentration (when dissolved in ink) was 10 μM, and then lyophilized. The micro vials each containing the probe DNA excluding moisture were used for the preparation of a DNA solution for spotting by the bubble jet method according to the following procedure.

[4] Discharge of DNA solution by BJ printer and immobilization on substrate surface Glycerol 7.0 wt%, ethylene glycol 5.0 wt%, hexanetriol 5.0 wt%, acetylenol EH (manufactured by Kawaken Fine Chemical Co., Ltd.) 1.0 wt % Aqueous solution was prepared. Subsequently, a predetermined amount of the mixed solvent was added to the microvials containing the eight types of probe DNAs (Table 1) prepared previously so that the DNA concentration was 10 μM at the final concentration (when the ink was dissolved). Thus, a DNA solution (ink) was prepared. The obtained DNA solution was filled in an ink tank for a bubble jet printer (trade name: BJF-850 manufactured by Canon Inc.) and mounted on a print head.

  The bubble jet printer has been modified so that printing on a flat plate is possible for use in spotting a DNA solution on the substrate surface. In addition, the print head of this bubble jet printer can spot a DNA solution droplet of about 5 picoliters at a pitch of about 120 micrometers by inputting a print pattern according to a predetermined file creation method. Yes.

  Subsequently, using the modified bubble jet printer, a spotting operation of each DNA solution was performed according to a predetermined printing pattern on a quartz glass substrate having a maleimide group introduced on the surface, thereby producing an array. . After confirming that the array-like spotting operation has been carried out reliably, it is allowed to stand in a humidified chamber for 30 minutes to react the maleimide group on the surface of the quartz glass substrate with the thiol group at the 5 ′ end of the probe DNA, thereby Was fixed.

[5] Washing After the reaction for 30 minutes, the DNA solution remaining on the substrate surface was washed away with 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl. A gene chip in which a plurality of types of single-stranded DNAs of interest were immobilized in an array on the surface of a quartz glass substrate was obtained.

<PCR amplification of sample DNA in genomic DNA extract>
PCR amplification of the 16s rRNA gene, which is the sample DNA, from the genomic DNA extract derived from the microorganism is performed according to the following procedures and conditions.

  First, PCR amplification reaction is performed using genomic DNA as a template, using the Forward Primer mixture containing 3 Forward Primers and the Reverse Primer mixture containing 3 Reverse Primers shown in Table 3 above. The PCR amplification reaction was performed using a commercially available PCR kit (TAKARA ExTaq), and the composition of the reaction solution is shown in Table 4.

  On the other hand, according to the temperature condition of PCR reaction, amplification reaction was performed using a commercially available thermal cycler according to the protocol shown in Table 4 below.

After the PCR amplification reaction was completed, the primer was removed using a commercially available PCR product purification column (QIAGEN QIAquick PCR Purification Kit), and the PCR amplification product was recovered. At that time, the obtained PCR amplification product was quantified.

<Preparation of labeled DNA strand using PCR amplification product as template>
As a fluorescent label, a labeled DNA strand was prepared according to the following procedure and conditions using a labeled oligonucleotide in which Cy3 was bound to the 5 ′ end.

  After the synthesis of the nucleotide chain, the fluorescently labeled compound Cy3 was covalently bound to the 5 'end of the nucleotide chain according to a conventional method. Thereafter, the labeled oligonucleotide was purified using HPLC. Table 5 shows the base sequence of the synthesized labeled oligonucleotide and its binding site (the number of bases from the 3 'end of the 16s rRNA gene derived from E. coli).

  Using the PCR amplification product obtained by the PCR amplification reaction as a template and a reverse primer, using a mixture of nine kinds of labeled oligonucleotides having the base sequences shown in Table 5, a complementary DNA chain extension reaction was carried out as follows. Follow the procedure and conditions of

  The extension reaction of the DNA strand was carried out in the form of a single-stranded PCR reaction using a commercially available PCR kit (TAKARA ExTaq). The reaction solution composition is shown in Table 6.

  On the other hand, the temperature condition of this extension reaction was carried out using a commercially available thermal cycler according to the protocol shown in Table 6 below.

After the completion of the DNA chain extension reaction, the primer is removed using a commercially available column for PCR product purification (QIAGEN QIAquick PCR Purification Kit), and a fluorescently labeled Cy3 derived from a labeled oligonucleotide is added to the 5 ′ end. The product was recovered. At that time, the obtained amplification product was quantified.

<Detection of sample DNA by hybridization method>
Using the gene chip prepared in <Preparation of DNA microarray> and the labeled sample DNA prepared in <Amplification and labeling of specimen (PCR amplification & incorporation of fluorescent label)>, a hybridization reaction is performed, and fluorescence labeling is performed. Was used to detect the hybrids formed.

(Gene chip blocking)
BSA (bovine serum albumin Fraction V: manufactured by Sigma) is added to 100 mM NaCl / 10 mM Phosphate Buffer to a concentration of 1 wt% to prepare a BSA solution. The gene chip prepared in <Preparation of DNA microarray> was immersed in this BSA solution at room temperature for 2 hours, and the quartz glass substrate surface of the gene chip was subjected to blocking treatment with BSA. After completion of the blocking treatment, 2 × SSC solution (NaCl 300 mM containing 0.1 wt% SDS (sodium dodecyl sulfate), Sodium Citrate (trisodium citrate dihydrate , Na 3 C 6 H 5 O 7 · 2H 2 O) 30mM, pH 7.0 The BSA solution remaining on the quartz glass substrate surface of the gene chip was washed. Next, after rinsing with pure water, the gene chip surface was drained with a spin dryer.

(Hybridization reaction)
The gene chip subjected to the blocking treatment was set in a hybridization apparatus (Genomic Solutions Inc. Hybridization Station), and a hybridization reaction between the labeled sample DNA and the probe DNA was performed according to the following procedures and conditions.

  The composition and conditions of the used hybridization solution are shown below.

[Hybridization solution]
A solution prepared by dissolving the purified labeled sample DNA prepared in <Amplification and labeling of sample (PCR amplification & incorporation of fluorescent label)> in 6 × SSPE buffer added with 10% HCONH ₂ was used.
6 × SSPE / 10% Form amide / Target (2 nd PCR Products total)
(6 × SSPE: NaCl 900 mM, NaH ₂ PO ₄ .H ₂ O 60 mM, EDTA 6 mM, pH 7.4)

[Hybridization conditions]
The hybridization reaction and the washing and drying operations after the reaction were performed under the conditions shown in Table 7.

<Detection of hybrid body (fluorescence measurement)>
After completion of the hybridization reaction, the fluorescence intensity derived from the fluorescence-labeled Cy3 is measured for each probe DNA spot on the spin-dried gene chip using a gene chip fluorescence detector (Axon, GenePix 4000B). It was.

  Table 8 shows the fluorescence intensity observed at the spot point of each probe DNA, and the binding site (approximately the number of bases from the 5 'end of the 16s rRNA gene) of the probe DNA.

  The labeled sample DNA sample used in the above hybridization reaction is a mixture of labeled DNA strands in which the DNA strand is extended from the base sequence of nine kinds of labeled oligonucleotides shown in Table 5 to the 3 ′ end. It is. As a result, with respect to the probe DNA; Pa-1 to Pa-8, one of the mixture of labeled DNA strands can form a hybrid with the probe DNA at a site close to its 5 ′ end. . In other words, at the spot points of probe DNA; Pa-1 to Pa-8, for each probe DNA, one of the mixture of labeled DNA strands forms a hybrid at a site close to its 5 ′ end. Are fixed. When a hybrid is formed at a site close to the 5 'end, fluorescence derived from the fluorescent label Cy3 added to the 5' end is observed with high efficiency. That is, for a plurality of types of labeled oligonucleotides used for the introduction of fluorescently labeled Cy3, it is possible to form a hybrid at the site where any of the plurality of types of probe DNAs is close to the 5 ′ end. By selecting in this way, in any of the plural types of probe DNAs, the fluorescence intensity derived from the fluorescent label Cy3 resulting from the formed hybrid is made uniform.

  In addition, a gene chip was prepared in the same procedure using probe DNAs; Hi-1 to Hi-8 shown in Table 2 derived from the Haemophilus influenza 16s rRNA gene. This gene chip was subjected to a blocking treatment and then subjected to a hybridization reaction using the labeled sample DNA sample. As a result, no fluorescence was detected due to the formation of hybrids with the probe DNAs; Hi-1 to Hi-8 shown in Table 2.

(Comparative Example 1)
In the step of <Preparation of labeled DNA strand using PCR amplification product as template> described in Example 1, only the labeled oligonucleotide of sequence 9 in Table 5 is used as the reverse primer used in the DNA strand extension reaction. Thus, a labeled DNA strand was prepared. Thereafter, the prepared labeled DNA strand was purified by the same procedure to obtain a labeled specimen DNA sample consisting of one kind of labeled DNA strand.

  Using the labeled sample DNA sample consisting of this single labeled DNA strand, a hybridization reaction was carried out with the probe DNA immobilized on the gene chip; Pa-1 to Pa-8. After completion of the hybridization reaction, the fluorescence intensity derived from the fluorescent label Cy3 is detected for the probe DNA; Pa-1 to Pa-8 spots on the gene chip using a gene chip fluorescence detector (Axon, GenePix 4000B). Measurements were made.

  Table 9 shows the fluorescence intensity observed at the spot points of each probe DNA, and the binding site of the probe DNA (approximately the number of bases from the 3 'end of the 16s rRNA gene).

  In contrast to the labeled DNA strand in which the DNA strand is extended to the 3 ′ end of the labeled oligonucleotide of the sequence 9, in the probe DNA; Pa-3 to Pa-8, fluorescence due to the formation of the hybrid is observed. Yes. In that case, the site | part to which probe DNA; Pa-3-Pa-8 couple | bonds with the labeled DNA strand which has the base sequence derived from the labeled oligonucleotide of this sequence 1 in the 5 'terminal. That is, the site where the probe DNA; Pa-8 to Pa-3 binds gradually from the 5 'end of the labeled DNA strand, and the measured fluorescence intensity decreases. As a result, the fluorescence intensity observed at the spot points of probe DNA; Pa-8 to Pa-3 capable of hybrid formation shows a large difference (fluorescence intensity variation) as shown in Table 9. Become.

  Of course, in the gene chip prepared using the probe DNAs shown in Table 2; Hi-1 to Hi-8, a labeled DNA strand in which the DNA strand is extended to the 3 ′ end of the labeled oligonucleotide of the sequence 9 Fluorescence due to hybrid formation was not observed.

(Example 2)
In the step of <Preparation of labeled DNA strand using PCR amplification product as template> described in Example 1, as a reverse primer used for DNA strand extension reaction, a mixture of nine types of labeled oligonucleotides shown in Table 5, As a Forward Primer, a Forward Primer mix described in Table 3 of Example 1 was added to prepare a labeled DNA strand. Thereafter, the prepared labeled DNA strand was purified by the same procedure to obtain a labeled specimen DNA sample comprising a plurality of types of labeled DNA strands.

  Using a labeled sample DNA sample composed of the labeled DNA strand, a hybridization reaction was performed with probe DNA immobilized on a gene chip; Pa-1 to Pa-8. After completion of the hybridization reaction, the fluorescence intensity derived from the fluorescent label Cy3 is detected for the probe DNA; Pa-1 to Pa-8 spots on the gene chip using a gene chip fluorescence detector (Axon, GenePix 4000B). Measurements were made.

  Table 10 shows the fluorescence intensities observed at the spot points of each probe DNA, and the binding sites (approximately the number of bases from the 3 'end of the 16s rRNA gene) of the probe DNA.

  As a result, with respect to the probe DNA; Pa-1 to Pa-8, one of the mixture of labeled DNA strands can form a hybrid with the probe DNA at a site close to its 5 ′ end. . In other words, at the spot points of probe DNA; Pa-1 to Pa-8, for each probe DNA, one of the mixture of labeled DNA strands forms a hybrid at a site close to its 5 ′ end. Are fixed. When a hybrid is formed at a site close to the 5 'end, fluorescence derived from the fluorescent label Cy3 added to the 5' end is observed with high efficiency. That is, for a plurality of types of labeled oligonucleotides used for the introduction of fluorescently labeled Cy3, it is possible to form a hybrid at the site where any of the plurality of types of probe DNAs is close to the 5 ′ end. By selecting in this way, in any of the plural types of probe DNAs, the fluorescence intensity derived from the fluorescent label Cy3 resulting from the formed hybrid is made uniform.

  In addition, a gene chip was prepared in the same procedure using probe DNAs; Hi-1 to Hi-8 shown in Table 2 derived from the Haemophilus influenza 16s rRNA gene. This gene chip was subjected to a blocking treatment and then subjected to a hybridization reaction using the labeled sample DNA sample. As a result, no fluorescence was detected due to the formation of hybrids with the probe DNAs; Hi-1 to Hi-8 shown in Table 2.

(Comparative Example 2)
In the step of <Preparation of labeled DNA strand using PCR amplification product as template> described in Comparative Example 1, as a reverse primer used in the DNA strand extension reaction, the labeled oligonucleotide of sequence 9 in Table 5 and Forward Primer are used. As described above, Forward Primer mix described in Table 3 of Example 1 was added to prepare a labeled DNA strand. Thereafter, the prepared labeled DNA strand was purified by the same procedure to obtain a labeled specimen DNA sample consisting of one kind of labeled DNA strand.

  Using the labeled sample DNA sample consisting of this single labeled DNA strand, a hybridization reaction was carried out with the probe DNA immobilized on the gene chip; Pa-1 to Pa-8. After completion of the hybridization reaction, the fluorescence intensity derived from the fluorescent label Cy3 is detected for the probe DNA; Pa-1 to Pa-8 spots on the gene chip using a gene chip fluorescence detector (Axon, GenePix 4000B). Measurements were made.

  Table 11 shows the fluorescence intensity observed at the spot point of each probe DNA, and the binding site of the probe DNA (approximately the number of bases from the 3 'end of the 16s rRNA gene).

  With respect to the labeled DNA strand in which the DNA strand is extended to the 3 ′ end of the labeled oligonucleotide of sequence 9, in the probe DNA; Pa-8 to Pa-5, fluorescence due to hybrid formation is observed. Yes. In that case, the site | part to which probe DNA; Pa-8-Pa-5 couple | bonds with respect to the labeled DNA strand which has the base sequence derived from the labeled oligonucleotide of this sequence 9 in the 5 'terminal. That is, the site where the probe DNA; Pa-8 to Pa-5 binds gradually from the 5 'end of the labeled DNA strand gradually decreases and the measured fluorescence intensity decreases. As a result, the fluorescence intensity observed at the spot points of probe DNA; Pa-8 to Pa-5 capable of hybrid formation shows a large difference (fluorescence intensity variation) as shown in Table 11. Become.

  Of course, in the gene chip prepared using the probe DNAs shown in Table 2; Hi-1 to Hi-8, a labeled DNA strand in which the DNA strand is extended to the 3 ′ end of the labeled oligonucleotide of the sequence 9 Fluorescence due to hybrid formation was not observed.

  In the nucleic acid labeling method according to the present invention, a labeled nucleic acid having a base sequence complementary to a partial region in the entire base sequence of the template nucleic acid is prepared from the template nucleic acid contained in the sample by an extension reaction. However, it can be suitably used when preparing a labeled nucleic acid sample for nucleic acid detection by probe hybridization reaction.

Claims (12)

A means for preparing a labeled nucleic acid for nucleic acid detection using a probe hybridization method,
Using a single-stranded nucleic acid prepared from a nucleic acid contained in a sample as a template nucleic acid,
Using a labeled oligonucleotide having a base sequence complementary to the partial base sequence contained in the base sequence of the template nucleic acid as a primer,
When a nucleic acid strand is extended to the 3 ′ end of the labeled oligonucleotide and an extension reaction is performed to prepare a nucleic acid inherent in the labeled oligonucleotide at the 5 ′ end to form a labeled nucleic acid. ,
As a primer for the extension reaction, using a primer set comprising at least two or more kinds of labeled oligonucleotides that differ in the complementary base sequences constituting the oligonucleotide,
With respect to a single-stranded template nucleic acid used for extension of a nucleic acid strand at the 3 ′ end of one labeled oligonucleotide contained in the primer set,
At least one of the other labeled oligonucleotides contained in the primer set has a base sequence complementary to the partial base sequence contained in the base sequence of the single-stranded template nucleic acid. A nucleic acid labeling method comprising: The nucleic acid labeling method according to claim 1, wherein the extension reaction is an asymmetric PCR reaction. The nucleic acid labeling method according to claim 1, wherein the extension reaction is a PCR reaction. The labeled oligonucleotide is:
The nucleic acid labeling method according to claim 2 or 3, wherein the oligonucleotide is labeled with one or more labels selected from the group consisting of a fluorescent substance and biotin. Nucleic acid detection using the probe hybridization method,
5. The nucleic acid labeling method according to claim 4, wherein a hybridization reaction for a DNA probe fixed or adsorbed on a solid surface is used. The DNA probe immobilized or adsorbed on the solid surface is
6. The method according to claim 5, wherein the method is in the form of a DNA chip or a DNA microarray. As the template nucleic acid,
The nucleic acid labeling method according to claim 1, wherein a single-stranded nucleic acid prepared as a PCR product from a nucleic acid contained in a specimen is used. The nucleic acid to be detected is
It is a 16s rRNA gene derived from bacteria, The nucleic acid labeling method as described in any one of Claims 1-7 characterized by the above-mentioned.   A labeled nucleic acid prepared by the nucleic acid labeling method according to claim 1.   A primer set comprising at least two or more kinds of labeled oligonucleotides that can be used in the nucleic acid labeling method according to claim 1. Based on the base sequence of the template nucleic acid,
A probe-corresponding site on the base sequence of the template nucleic acid, in which the base sequence of the probe DNA used for nucleic acid detection using the probe hybridization method is selected;
About at least two kinds of sites complementary to the primer on the base sequence of the template nucleic acid having a base sequence complementary to the base sequence of the at least two or more kinds of labeled oligonucleotides,
When defining the relative position of at least two kinds of complementary sites to the primer based on the number of bases up to the presence position of at least two kinds of complementary positions to the primer, using the probe corresponding site as a reference point,
The primer according to claim 10, wherein the base sequences of the at least two kinds of labeled oligonucleotides are selected so that at least two kinds of relative positions of the complementary sites to the primer are different from each other. ·set. As the at least two or more kinds of labeled oligonucleotides contained in the primer set,
Sequence 1 to Sequence 9 below:
Sequence 1; 5 'actgctgcctcccgtaggagtctgg 3'
Sequence 2; 5 'cgtattaccgcggctgctggcacg 3'
Sequence 3; 5 'gcgtggactaccagggtatctaatcctgtttg 3'
Sequence 4; 5 'tcgaattaaaccacatgctccaccgcttgtgcggg 3'
Sequence 5; 5 'ggtaaggttcttcgcgttgc 3'
Sequence 6; 5 'ttgacgtcatccccaccttcctcc 3'
Sequence 7; 5 'ccattgtagcacgtgtgtagccc 3'
Sequence 8; 5 'tggtgtgacgggcggtgtgtacaag 3'
Sequence 9; 5 'taccttgttacgacttcacccca 3'
12. The primer set according to claim 11, comprising a labeled oligonucleotide having at least two or more kinds of base sequences selected from the group consisting of: