JP2007244349A - Method for detecting bacteria of periodontal disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for efficiently and precisely detecting specific five kinds of bacteria of periodontal disease from a specimen such as saliva by an invader assay method. <P>SOLUTION: The quantitative detection is carried out by using as a target base the 748'th base of 16SrRNA gene of Porphyromonas gingivalis, the 191st base of the same gene of Prevotella intermedia, the 842nd base of the same gene of Actinobacillus actinomycetemcomitans, the 155th base of the same gene of Bacteroides forsythus or the 155th or 420th base of the same gene of Treponema denticola in the bacteria of the periodontal disease in the invader assay method. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for detecting a specific bacterium. Specifically, the present invention is an invention aimed at quantitatively detecting a specific periodontal disease bacteria using an invader assay method.

  Periodontal disease is known to be improved by proper brushing and removal of calculus at the stage of gingivitis. Periodontal disease is known to progress in the subgingival pocket due to colonization and proliferation of bacterial flora in the tooth-periodontal tissue. Periodontal disease is almost painless even if it progresses, and often progresses before it is noticed, but if left untreated, it may eventually lead to teeth falling out.

  Particularly in Japan in recent years, the westernization of eating habits has progressed, and the degree of suffering from periodontal diseases has increased due to the tendency to prefer relatively soft foods and increased mental stress.

  Countermeasures for periodontal disease include correct toothbrushing, plaque control through regular checkups, and preventive actions such as improving dietary habits, but in reality, how accurately periodontal diseases that are progressing are discovered, The most important point is how to treat the patient appropriately.

  Examples of diagnostic methods for periodontal diseases include X-ray examination, plaque adhesion examination, subgingival pocket measurement examination, and the like.

  Periodontal diseases are infections caused by periodontal pathogens, especially in severe chronic periodontitis and invasive periodontitis, there are many specific periodontal pathogens that are not inherently present in the oral cavity. It is considered that the cause-removal therapy can be performed by clarifying the treatment target by quantitatively detecting the periodontal disease bacteria.

  However, for example, it is known that more than 250 types of bacteria are present in the bacterial flora below the gingival margin, and it is difficult to identify and quantitatively detect periodontal disease bacteria from these bacteria.

  In the present invention, in view of such a situation, Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), which is known as a causative agent of periodontal disease, from a sample such as saliva, which can contain many types of bacteria. The object is to provide a means for efficiently and reliably quantitatively detecting Actinobacillus actinomycetemcomitans (Aa), Bacteroides forsythus (Bf) and Treponema denticola (Td).

  In order to solve the above problems, the present inventor performed quantitative detection of periodontal disease bacteria by an invader assay method, that is, the following nucleotide chains (1) to (3):

(1) a template nucleotide chain having a target base to be detected;

(2) a first complementary strand having a base adjacent to the target base of the template nucleotide chain and capable of hybridizing from the adjacent base toward the 3 'side of the template nucleotide chain;

(3) having a base complementary to the target base, capable of hybridizing from the complementary base toward the 5 ′ side of the template nucleotide chain, and unable to hybridize toward the 3 ′ side, Two complementary strands;
To form a partial triplex structure in the target base portion of the template nucleotide chain (1), and then the 3 ′ side of the second complementary strand (3) that has formed the triplex structure The target base of the template nucleotide chain (1) is detected by detecting a nucleotide chain on the non-hybridizing side of the second complementary chain (3) released by the enzyme reaction by acting a restriction enzyme that specifically cleaves. Aiming to be performed by a detection method (hereinafter, unless otherwise specified, this detection method is called an invader assay method), a specific detection target base, the first complementary strand, the second complementary strand, etc. After examination, the present invention was completed.

    That is, the present invention relates to an invader assay method of periodontopathic bacteria of the group consisting of Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Actinobacillus actinomycetemcomitans (Aa), Bacteroides forsythus (Bf), and Treponema denticola (Td). 1 to 5 types of genes encoding 16S rRNA selected from the above-mentioned template nucleotide chain (1), the following (a) to (e) in the nucleotide chain:

(A) the base at position 748 of the gene encoding Pg 16S rRNA (including both complementary base sequences);
(B) the 191st base of a gene (including complementary base sequences) encoding Pi 16S rRNA;
(C) the 842nd base of the gene encoding 16S rRNA of Aa (including both complementary base sequences);
(D) the 842nd base of a gene (including complementary base sequences) encoding Bf 16S rRNA;
(E) the 155th base or the 420th base of a gene (including complementary base sequences) encoding Td 16S rRNA;
The present invention provides a method for detecting periodontal pathogens (hereinafter also referred to as the present detection method), characterized in that quantitative detection of these periodontal pathogens is performed by detecting as a target base.

  In the present invention, “complementary” means that the nucleic acid has a degree of complementarity capable of hybridizing to a nucleic acid having a certain base sequence under conditions that are stringent (56 to 68 ° C., in the presence of 50 mM or more sodium ions). It means a base sequence. In addition, the “both complementary base sequences” refers to a base sequence including both a complementary base sequence having a −strand relationship to the + strand and / or a complementary base sequence having a + strand relationship to the − strand. Meaning.

  The present invention provides a means for efficiently and accurately detecting a specific periodontal disease bacteria in a sample such as saliva.

[Invader assay method]
FIG. 1 is a diagram showing an overview of an invader assay method [Third Wave Technologies (USA)].

  In FIG. 1, a first complementary strand 12 is hybridized to a template nucleotide strand (detection target gene) 11.

  The first complementary strand 12 is a template in which the base [A (adenine) in this figure] complementary to the detection target base [T (thymine) in this figure] in the template nucleotide chain 11 is located at the 3 ′ end. A nucleotide chain complementary to the nucleotide chain 11 (in this example, the base at the 3 ′ end of the first complementary chain 12 is complementary to the base to be detected, but the base is complementary. Otherwise, the base may interfere with the association reaction between the base to be detected and the second complementary strand described below to form a partial triplex structure).

  Next, the second complementary strand 13 is further hybridized to the partial double strand of the template nucleotide strand 11 and the first complementary strand 12.

  The second complementary strand 13 has a “complementary portion” 131 complementary to the template nucleotide strand 11 on the 3 ′ side, and is continuous with the template nucleotide strand 13 provided with a detection element. The detection portion 132 that does not hybridize is a complex nucleotide chain on the 5 ′ side, and the most 5 ′ base of the complementary portion 131 is a base complementary to the detection target base (T) ( A).

  By this second hybridization reaction, the detection target base portion (T) of the template nucleotide chain 11 is the most 5 ′ of the 3 ′ terminal base of the first complementary strand 12 and the complementary portion 131 of the second complementary strand. A partial triple chain structure with the base (A) on the side will be formed.

  Next, the partial triple-stranded structure is allowed to act on the 3 ′ side thereof with a nuclease 14 having an activity of specifically cleaving, so that the detection portion 132 ′ of the second complementary strand 13 cleaved by the nuclease [ By detecting the 3 ′ end is a base (A) complementary to the detection target base (T)], it can be clarified that the template nucleotide chain 11 is the detection target gene. .

  In this figure, a fluorescent dye 151 near the 5 'end and a hairpin probe (nucleotide chain) 15 labeled with a fluorescence quencher (quencher) 152 near the 3' end coexist with the hybridizing system. By doing so, it is possible to detect the detection target gene.

  In the present invention, a hairpin probe is expressed as “one nucleotide chain is bent and hybridized with the 3 ′ end and the 5 ′ end as both ends, and a part of the nucleotide chain on the 3 ′ end side is hybridized. (1) The protruding portion on the 3 ′ end side can hybridize with the above-mentioned free nucleotide chain, and is adjacent to the most 5′-side base of the protruding portion. The matching 5′-side base is the target base, and (2) a probe in which a fluorescent dye is labeled in the vicinity of the 5 ′ end and a fluorescent quencher is labeled in the vicinity of the 3 ′ side of the fluorescent dye. It is prescribed.

  The single-stranded portion 3 ′ side of the hairpin probe 15 is designed to be complementary to the detection portion 132 of the second complementary strand 13 and is the 5′-most portion of the single-stranded portion. A further base on the 5 ′ side adjacent to the base is a detection target base (T). When the detection portion 132 ′ is hybridized with the single-stranded portion of the hairpin probe 15, the base (A) at the 3 ′ end of the detection portion 132 ′ is the tip of the double-stranded portion of the hairpin probe 15. Again, a partial triple chain structure is formed. On the other hand, the nuclease 14 acts again, and the nucleotide chain between the fluorescent dye 151 and the fluorescence quenching substance 152 in the hairpin probe 15 is cleaved, so that the fluorescent dye 151 is released, and the fluorescence quenching substance 152 It is released from the fluorescence quenching reaction, and the original fluorescence can be detected. By detecting this fluorescence, it can be clarified that the template nucleotide 11 is a detection target gene.

  On the other hand, when the base corresponding to the detection target base of the template nucleotide 11 is a base other than the detection target base [eg, G (guanine)], at least for detection in the reaction system similar to the above. The 3 ′ terminal base (A) of the portion 132 is not complementary to the other base. Therefore, since the partial triplex structure as described above is not formed, the detection portion 132 is not cleaved from the second complementary strand by the nuclease 14, and the free detection portion 132 ′ does not appear in the system. Become. Therefore, the hairpin probe 15 also maintains the state in which the fluorescent dye 151 and the fluorescence quencher 152 are close to each other, and substantial fluorescence is not recognized. In this case, it becomes clear that the template nucleotide 11 is not a detection target gene.

  In this manner, the detection target gene can be identified using the invader method.

  In addition, although the detection method using the hairpin type probe is illustrated here, for example, a label such as a fluorescent label or an isotope label is provided on the detection portion 132 and the label is counted. The detection target gene can be detected.

  Quantification of the detection target gene by the invader method can be generally performed according to the following principle.

The reaction at the stage of forming the partial triple chain structure (primary reaction) is as follows:
[A] = α1 × [T] × t
(In the formula, [] represents the concentration in the system, A represents the amount of the free detection moiety 132 ′ (flap), α1 represents the rate of cleavage in the partial triple chain structure in the primary reaction, and T Represents the amount of the sample gene, and t represents the elapsed reaction time).

Here, when the above equation is differentiated with respect to time t to obtain the rate of change per hour of the flap amount in the system,
d [A] / t = α1 × [T]
It is represented by

Next, the complementary strand binding is performed so that the flap 132 ′ forms a partial triplex structure in the hairpin type probe 15, whereby the nucleotide chain between the fluorescent dye 151 and the fluorescence quencher 152 is cleaved and released. The reaction (secondary reaction) in which the fluorescent dye part is released into the system is
[S] = α2 × [A] × t + β1 × [X] × t
(In the formula, S represents the amount of the secondary reaction product (expressed by the amount of fluorescence by the free fluorescent dye 151), α2 represents the rate of cleavage in the partial triple chain structure in the secondary reaction, and t represents the reaction. Elapsed time, β1 represents the non-specific cleavage rate, and X represents the amount of non-specific product.

When this is differentiated by time t in the same manner as described above to determine the rate of change in the amount of secondary reaction products,
d [S] / t = α2 × [A] + β1 × [X]
It is represented by

In the system, the primary and secondary reactions proceed macroscopically in parallel,
d [S] / t = α2 × [A] + β1 × [X]
d [S] / t = α2 × α1 × [T] × t + β1 × [X]
d [S] / t = α1α2 [T] t + β1 [X]
It is represented by If this is integrated with t,
[S] = 1 / 2α1α2 [T] t ² + β1 [X] t

Here, β1 [X] t can be regarded as constant regardless of the amount of the specimen gene.
The amount of fluorescence S indicating the amount of secondary reaction products is:
[S] = At ² + B (where A and B are constants)
It is represented by

Therefore, when the desired quantitative value of the target gene to be detected is Y for the fluorescence value and X for the reaction time,
It is expressed as A in Y = AX ² + B (secondary equation).

  The quantitative process by the above invader assay method has already been reported in the literature (Jeff et al, PNAS, vol. 97, 8272-8277, 2000).

[This detection method]
Periodontopathic bacteria that are the targets of quantitative detection (hereinafter referred to as “detection” unless otherwise specified) of the present invention are Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Actinobacillus actinomycetemcomitans (Aa), Bacteroides, as described above. forsythus (Bf) and Treponema denticola (Td). In this detection method, the gene containing the target to be detected is a gene encoding 16SrRNA of each periodontal disease bacterium (hereinafter also referred to as 16SrRNA gene). The 16S rRNA gene of Pg is as shown in SEQ ID NO: 1 and FIG. 2, the 16S rRNA gene of Pi is as shown in SEQ ID NO: 2 and FIG. 2, and the 16S rRNA gene of Aa is shown in SEQ ID NO: 3 and FIG. The 16S rRNA gene of Bf is as shown in SEQ ID NO: 4 and FIG. 2, and the 16S rRNA gene of Td is as shown in SEQ ID NO: 5 and FIG.

  Samples to which this detection method should be applied are all those for which periodontal disease bacteria should be detected. Usually, it is a saliva sample or a plaque sample, but blood samples such as serum and plasma, and urine samples can also be used to identify the causative bacteria of severe periodontal diseases.

  A method for preparing a gene from these specimens can be performed according to a conventional method. In general, a specimen to be used, that is, plaques on saliva or paper points can be obtained by extracting DNA in the specimen by an ordinary method such as SDS method, phenol method, ethanol method, or automatic DNA extractor. . The desired periodontal disease bacteria are obtained by performing the above-described invader assay for the nucleic acid sample thus obtained (specimen-derived DNA) with the target base, the first complementary strand, and the second complementary strand determined. Can be quantitatively detected. In addition, although a detection parameter | index is not specifically limited, The hairpin type probe mentioned above is suitable.

( Target base )
In the 16S rRNA gene of these periodontal disease bacteria, the base that is the target base of this detection method is
(A) The 748th base of Pg 16S rRNA gene (including both complementary base sequences), specifically, guanine or cytosine.
(B) The 191st base of Pi's 16S rRNA gene (including complementary both base sequences), specifically, guanine or cytosine.
(C) The 842st base of Aa 16S rRNA gene (including complementary both base sequences), specifically, guanine or cytosine.
(D) The 842th base of Bf 16S rRNA gene (including complementary base sequences), specifically, thymine or adenine.
(E) The 155th base of the Td 16S rRNA gene (including both complementary base sequences), specifically thymine or adenine, or the 420th base, specifically thymine or adenine.

( First complementary strand )
In this detection method, it is necessary to use the first complementary strand as in the above example in order to detect the target base. The first complementary strand is a nucleotide chain (including complementary both base sequences) consisting of a base sequence of at least 9 bases from the target base (not including the base) toward the 3 ′ side of the detection target gene. The base chain that is complementary to the target base of the detection target gene, and the total number of bases of the first complementary chain is preferably about 10 to 50 bases, Particularly preferred is about 20 to 45 bases.

  Specific examples of the first complementary strand include the following nucleotide strands. In the following nucleotide chain, N at the 5 ′ end (indicating that any nucleobase such as G or C or A or T can be selected) is the above “base adjacent to the target base”.

(A) For the Pg 16S rRNA gene (including both complementary nucleotide sequences), the nucleotide sequence represented by SEQ ID NO: 6 (including both complementary nucleotide sequences) can be mentioned.
5′-CCCACGCCTTCGTGCTTCAGTGTCAGTN-3 ′ (SEQ ID NO: 6)

(B) For the Pi 16S rRNA gene (including complementary both nucleotide sequences), the nucleotide sequence represented by SEQ ID NO: 7 (including both complementary nucleotide sequences) can be mentioned.
5′-CAACAAGCTAATCAGACGCATCCCCATCCTCCACN-3 ′ (SEQ ID NO: 7)

(C) For the Aa 16S rRNA gene (including complementary both nucleotide sequences), the nucleotide sequence represented by SEQ ID NO: 8 (including both complementary nucleotide sequences) can be mentioned.
5′-GTCGATTTATCACGTTAGCTTCGGGCACCAGGGN-3 ′ (SEQ ID NO: 8)

(D) For the Bf 16S rRNA gene (including both complementary nucleotide sequences), the nucleotide sequence represented by SEQ ID NO: 9 (including both complementary nucleotide sequences) can be mentioned.
5′-CCCAGGTGGATTACTTAACGCTTTCGCTGTAGAGCTTACN-3 ′ (SEQ ID NO: 9)

(E) For Td 16S rRNA gene (including complementary both base sequences), SEQ ID NO: 10 (when the 155th base of the gene is the target base), or SEQ ID NO: 11 (420th of the gene And a base sequence (including both complementary base sequences) represented by a target base).
5′-CGGAGCCGTAGCTCCTTTCCTCATTTACCTTTN-3 ′ (SEQ ID NO: 10)
5′-CATGACTACCGTCATCAAAGAAGCATTCCCTCTTCTTCTTN-3 ′ (SEQ ID NO: 11)

( Second complementary strand )
In this detection method, in order to detect the target base, it is necessary to use the second complementary strand together with the first complementary strand as in the above example. The second complementary strand is non-complementary (non-hybridized) 5′-side nucleotide strand (corresponding to “detection portion 132” in the above example) and complementary (target base). Is a complex nucleotide chain comprising a 3′-side nucleotide chain (corresponding to “complementary portion 131” in the above example) containing a base complementary to and hybridizing with the detection target gene. The total number of bases in the complementary hybridizing portion of the second nucleotide chain is preferably about 10 to 30 bases, and particularly preferably about 12 to 25 bases. Any non-complementary nucleotide chain can be used, and preferably a nucleotide chain of about 7 to 30 bases, particularly preferably about 10 to 20 bases.

  Specific examples of the second complementary strand include the following nucleotide strands. In the following example, X represents a non-complementary nucleotide chain portion.

(A) For the Pg 16S rRNA gene (including complementary both nucleotide sequences), the nucleotide sequence represented by SEQ ID NO: 12 (including both complementary nucleotide sequences) can be mentioned.
5′-XCGCAGTATGGCAAGC-3 ′ (SEQ ID NO: 12 except for nucleotide chain X)

(B) For the Pi 16S rRNA gene (including complementary both nucleotide sequences), the nucleotide sequence represented by SEQ ID NO: 13 (including both complementary nucleotide sequences) can be mentioned.
5′-CGATGAATCTTTGGTCCA-3 ′ (SEQ ID NO: 13)

(C) For the Aa 16S rRNA gene (including both complementary nucleotide sequences), the nucleotide sequence represented by SEQ ID NO: 14 (including both complementary nucleotide sequences) can be mentioned.
5′-XCTAAACCCCAATCCCCA-3 ′ (SEQ ID NO: 14 except for nucleotide chain X)

(D) For the Bf 16S rRNA gene (including complementary both nucleotide sequences), the nucleotide sequence represented by SEQ ID NO: 15 (including both complementary nucleotide sequences) can be mentioned.
5′-XACTATATCGCAAACTCCTAG-3 ′ (SEQ ID NO: 15 except for nucleotide chain X)

(E) For a gene encoding Td 16S rRNA (including complementary both base sequences), SEQ ID NO: 16 (when the 155th base of the gene is the target base) or SEQ ID NO: 17 (of the gene And a base sequence (including complementary both base sequences) represented by the 420th base is a target base).

5′-ATGTAAATGAGCACATTCG-3 ′ (SEQ ID NO: 16)
5′-XATTCTTCATCTGCAAAAGAATT-3 ′ (SEQ ID NO: 17 except for nucleotide chain X)

( Total number of oral bacteria )
In this detection method, it is possible to determine the periodontal disease state of the subject more accurately by calculating the total number of bacterial cells in the sample and determining the ratio of specific periodontal bacteria in the sample. It is. That is, particularly when saliva is used as a specimen, the concentration of bacteria contained in the saliva tends to vary depending on the method of collecting saliva. For example, when comparing saliva naturally secreted into the oral cavity with saliva obtained by chewing gum and promoting secretion, the latter saliva has fewer cells per saliva. It is normal. Therefore, in order to quantify the number of periodontal disease bacteria, first, the total number of cells is measured based on the nucleic acid extracted from saliva, and the number of cells per unit volume of oral bacteria in the saliva sample is confirmed. Quantitative detection of periodontal disease bacteria by measuring the periodontal disease bacteria and determining the ratio of the number of periodontal disease bacteria to the total number of oral bacteria. Is possible. The method for calculating the total number of bacterial cells in a sample follows a conventional method, but an embodiment using an invader assay method can be exemplified as a suitable method as in the present detection method.

  In this case, the target bases to be detected by the invader assay method are the base 1382 of Aa 16S rRNA gene, the base 1384 of Pg, the base 1350 of Pi, the base 1341 of Bf, and the base 1353 of Td. (Complementary base, specifically including guanine or cytosine), and the first complementary strand capable of commonly detecting the target bases of these different periodontal pathogens And the second complementary strand are selected, an invader assay method is performed, and quantitative detection is performed as described above, whereby the desired number of oral bacteria can be calculated.

In this case, for example, the first complementary strand is
As 5′-TGACGGGCGGTGTGTACAAGGCN-3 ′ (SEQ ID NO: 18), and
A second complementary strand,
5′-XCCGGGAACGTATTCACC-3 ′ (SEQ ID NO: 19 except for nucleotide chain X)
Thus, the number of oral bacteria can be determined by performing an invader assay on the sample-derived DNA.

[Detection kit]
In the present invention, a kit for detecting the above five specific periodontal pathogens (hereinafter also referred to as the present detection kit) for carrying out the present detection method is also provided.

The present detection kit includes the first complementary strand and the second complementary strand defined as described above, which are used for performing the invader assay method in the present detection method, as components of the kit. That is, the target base is the one shown in the above-mentioned "target base" section, the first complementary strand is the one shown in the above-mentioned "first complementary strand" section, and the second complementary strand is As shown in the above-mentioned “second complementary strand” section, it is contained in this detection kit.
Preferably, the detection kit contains a detection means typified by the above-described hairpin probe and a restriction enzyme having an activity of specifically cleaving the 3 ′ side of the partial triplex structure. be able to.

Furthermore, the buffer used for performing an invader assay method and the tube and plate for a test can also be contained.
It is also preferable to add a first complementary strand and a second complementary strand as components of the kit for performing an invader assay for the purpose of calculating the total number of cells in the specimen.

  Examples of the present invention will be disclosed below. However, the scope of the present invention is not limited by this embodiment.

[Detection of periodontal disease bacteria]
(1) Detection object As described above, detection objects include Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Actinobacillus actinomycetemcomitans (Aa), Bacteroides forsythus (Bf), and Treponema denticola (Td). It is.

(2) Detection method Detection by the invader assay method was performed as follows.
As reagents (reaction buffer, cleavase XI, etc.), Invader core reagent kit (the above-mentioned hairpin probe was used as a probe) supplied from third wave technologies was used. To the 96-well plate, add 3.5 μl of cleavase XI FRET mix, 1.0 μl of Cleavase XI Enzyme / MgCl2 solution and 100 μΜ FRET probe 0.105 μl, 10 μM invader oligo 0.105 μl, 2.79 μl of 10 mM MOPS buffer, 2.5 μl of DW, and sample (standard reagent) And 5.0 μl of the sample-derived DNA solution), reacted at 63 ° C. or 65 ° C. for 4 hours, and measured the fluorescence value every 120 seconds. The amplification factor of the fluorescence value obtained with time from the reaction tube was converted into a quadratic curve based on the literature (Jeff et al, PNAS, vol. 97, 8272-8277, 2000). Using the slope of this curve as the measurement value of each reaction tube, a calibration curve was obtained from the measurement value of the standard reagent, and the quantitative value of the target gene was calculated from the measurement value of the test sample.

(3) Total number of bacterial cells in the oral cavity As described above, grasping the total number of bacterial cells in the oral cavity of the subject is an accurate evaluation for periodontal diseases, particularly when using a saliva sample. It is an important element. In this example, the total number of cells in the sample, the target base in the invader assay method, the 1382th of the Aa 16S rRNA gene, the 1384th of the Pg, the 1350th of the Pi, the 1341st of the Bf, and , And the 1353rd base of Td (specifically, cytosine: see FIG. 2). In this example, the first complementary strand that uses this total number of bacteria in the invader assay method,
As 5′-TGACGGGCGGTGTGTACAAGGCA-3 ′ (SEQ ID NO: 20), and
A second complementary strand,
As 5′-CGCGCCGAGGCCGGGAACGTATTCACC-3 ′ (SEQ ID NO: 21), detection was performed by the invader assay method as described in (2) above.
(When this pair of complementary strands is used, the hybridization reaction by the invader assay method is performed in the region of the underlined portion 1 of the five types of periodontal disease bacteria 16S rRNA genes in FIG. 2, and the target base 11 is detected. .)

  The measured values in this example were calculated as the number of periodontal bacteria / total number of oral bacteria × 100 = the ratio (%) of periodontal bacteria to the whole oral bacteria.

(4) Detection of specific periodontal bacteria (a) Detection of Porphyromonas gingivalis (Pg)
390, 424, 428, 433, 528, 567, 587, 622, 626, 666, 711, 804, 816 in the 16S rRNA gene sequence of Pg bacteria (Gene bank accetion number: X73964, SEQ ID NO: 1, FIG. 2) The bases 973, 980, and 1077 are used as target bases that are recognized as cleavage points by the Cleavase enzyme (a restriction enzyme that specifically cleaves the 3 ′ side of the partial triple-stranded structure). A second pair of complementary strands was designed, and the detection sensitivity and specificity were examined using these. As a standard reagent for quantification, DNA was purified from Pg strain (ATCC33277), and the entire 16S rRNA gene sequence was PCR amplified and incorporated into a plasmid (pCRII-TOPO: Invitrogen), and this plasmid was used for detection sensitivity test. Plasmids were prepared in 10 3 to 10 7 7 copy / measurement tubes and used as standard reagents. In the detection sensitivity test, when the 390th, 424th, 428th, 587th, 666th, 711th, and 816th bases are set as the target bases in the above-described 16 sets of complementary strands of the invader assay method, the cube of 10 A measurement range of -10 to the seventh power copy / measurement tube was obtained. Specificity tests were performed on these seven sets of complementary strands. Saliva was selected as a sample for the specificity test. For DNA, 200 μl of saliva was extracted into 100 μl of eluate using MagNA Pure LC (Roche) and DNA Isolation kit I (Roche), treated at 95 ° C. for 5 minutes, and 5 μl was measured. used. Ten examples of DNA samples that became positive or negative by the real-time PCR method using the 615th to 682nd bases of the 16S rRNA gene of Pg bacteria as the gene amplification region were used for the above seven sets of complementary strands. As a result of measurement by the invader assay method, a pair of complementary strands in which a difference from the result in the real-time PCR method was not recognized was a group having the following base 711 (guanine) as a target base:

First complementary strand: 5′-CCCACGCCTTCGTGCTTCAGTGTCAGTT-3 ′ (SEQ ID NO: 22)
Second complementary strand: 5′-CGCGCCGAGGCGCAGTATGGCAAGC-3 ′ (SEQ ID NO: 23)
(When this set of complementary strands is used, a hybridization reaction is carried out by the invader assay method in the region of the underlined portion 2 of the 16S rRNA gene of P. g. In FIG. 2, and the target base 21 is detected. )

It was only. Furthermore, when the invader assay method using this pair of complementary strands was detected, 64 positive samples obtained by the real-time PCR method were compared with the real-time PCR method. A good correlation with a coefficient of 0.9109 was obtained.
In addition, detection by an invader assay method using a pair of complementary strands with the 711st base as a target base was performed using Pg, Pi, Bf, Aa, Td, Neisseria subflava, Neisseria sicca, As a result of measuring 100 ng of DNA extracted from Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Streptococcus mitis, Streptococcus anguinosus, Streptococcus gordanii, Streptococcus sanguis, it was found that only Pg bacteria can be detected specifically. In addition, P. which became positive by this detection method. g. The ratio in the total number of oral cells of bacteria was 0.007 to 0.177%.

(B) Detection of Prevotella intermedia (Pi)
PiS 16S rRNA gene sequence (Gene bank accetion number: X73964, SEQ ID NO: 2, Fig. 2), the 138, 180, 191, 413, 423, 433, and 552th bases were replaced with the Cleavase enzyme (partial triple-stranded structure). As a target base that is recognized as a cleavage point by a restriction enzyme that specifically cleaves the 3 ′ side of the DNA, seven types of first and second complementary strand pairs are designed, and these are used to detect detection sensitivity and Specificity was examined. As a standard reagent for quantification, DNA was purified from Pi strain (ATCC49046), and the entire 16S rRNA gene sequence was amplified by PCR and then incorporated into a plasmid (pCRII-TOPO: Invitrogen), and this plasmid was used for detection sensitivity test. Plasmids were prepared in 10 3 to 10 7 7 copy / measurement tubes and used as standard reagents. In the detection sensitivity test, when the 180th, 191st, and 423rd bases are used as the target base in the set of complementary strands of the above seven methods of invader assay, 10 3 to 10 7 copies / measurement The measurement range of the tube was obtained. Specificity tests were performed on these three sets of complementary strands. Saliva was selected as a sample for the specificity test. For DNA, 200 μl of saliva was extracted into 100 μl of eluate using MagNA Pure LC (Roche) and DNA Isolation kit I (Roche), treated at 95 ° C. for 5 minutes, and 5 μl was measured. used. Ten cases each of positive or negative DNA samples by the real-time PCR method using the 91st to 191st bases of PiS 16S rRNA gene as the gene amplification region were used with the above 7 sets of complementary strands. As a result of measurement by the invader assay method, a pair of complementary strands in which a difference from the result in the Real-time PCR method was not recognized was a group having the 191st base (guanine) as a target base:

First complementary strand: 5′-CAACAAGCTAATCAGACGCATCCCCATCCTCCACA-3 ′ (SEQ ID NO: 24)
Second complementary strand: 5′-CGCGCCGAGGCGATGAATCTTTCGTCCA-3 ′ (SEQ ID NO: 25)
(When this set of complementary strands is used, a hybridization reaction is carried out by the invader assay method in the region of the underlined portion 3 of the 16S rRNA gene of Pi bacteria in FIG. 2, and the target base 31 is detected. )

  It was only. Furthermore, when the invader assay method using this pair of complementary strands was detected, 64 positive samples obtained by the real-time PCR method were compared with the real-time PCR method. A good correlation was obtained with a coefficient of 0.9623.

  In addition, detection by an invader assay method using a pair of complementary strands with the 191st base as a target base was used for Pg, Pi, Bf, Aa, Td, Neisseria subflava, Neisseria sicca, As a result of measuring 100 ng of DNA extracted from Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Streptococcus mitis, Streptococcus anguinosus, Streptococcus gordanii, Streptococcus sanguis strains, it was found that only Pi bacteria can be specifically detected. In addition, P. which became positive by this detection method. i. The ratio of the number of bacteria in the total number of oral cells was 0.011 to 1.090%.

(C) Detection of Actinobacillus actinomycetemcomitans (Aa)
151A, 153, 186, 315, 338, 451, 467, 586, 588, 590, 592, 594, 597 in the 16S rRNA gene sequence of Aa bacteria (Gene bank accetion number: M75037, SEQ ID NO: 3, FIG. 2) 608, 659, 744, 837, 842, and 853rd bases, 20 types of target bases that Cleavase enzyme (a restriction enzyme that specifically cleaves the 3 ′ side of the partial triplex structure) recognizes as a cleavage point. A set of a first complementary strand and a second complementary strand was designed, and the detection sensitivity and specificity were examined using these. As a standard reagent for quantification, DNA was purified from Aa strain (ATCC43718), and the entire 16S rRNA gene sequence was PCR amplified and incorporated into a plasmid (pCRII-TOPO: Invitrogen), and this plasmid was used for detection sensitivity test. Plasmids were prepared in 10 3 to 10 7 7 copy / measurement tubes and used as standard reagents. In the detection sensitivity test, when the bases 151, 153, 186, 315, 451, 467, 586, 837, 842, and 853 are used as target bases in the above-described 20 sets of complementary strands of the invader assay method A measurement range of 10 3 to 10 7 copies / measurement tubes was obtained. Specificity tests were performed on these 10 sets of complementary strands. Saliva was selected as a sample for the specificity test. For DNA, 200 μl of saliva was extracted into 100 μl of eluate using MagNA Pure LC (Roche) and DNA Isolation kit I (Roche), treated at 95 ° C. for 5 minutes, and 5 μl was measured. used. Ten examples of DNA samples that were either positive or negative by Real-time PCR using the 465th to 609th bases of the 16S rRNA gene of Aa as a gene amplification region were used for the above 10 sets of complementary strands. As a result of measurement by the invader assay method, a pair of complementary strands in which a difference from the result in the real-time PCR method was not recognized was a group having the 842nd base (guanine) as a target base:

First complementary strand: 5′-GTCGATTTATCACGTTAGCTTCGGGCACCAGGGT-3 ′ (SEQ ID NO: 26)
Second complementary strand: 5′-CGCGCCGAGGCTAAACCCCAATCCCCA-3 ′ (SEQ ID NO: 27)
(When this pair of complementary strands is used, a hybridization reaction is carried out by the invader assay method in the region of the underlined portion 4 of the 16S rRNA gene of Aa bacteria in FIG. 2, and the target base 41 is detected.)

  It was only. In addition, detection by invader assay using this pair of complementary strands was compared with quantitative analysis of 40 positive samples by real-time PCR method with real-time PCR method. A good correlation was obtained with a coefficient of 0.8013.

  In addition, detection by an invader assay method using a pair of complementary strands with the 842nd base as a target base was performed using Pg, Pi, Bf, Aa, Td, Neisseria subflava, Neisseria sicca, As a result of measuring 100 ng of DNA extracted from strains of Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Streptococcus mitis, Streptococcus anguinosus, Streptococcus gordanii, Streptococcus sanguis, it was found that only Aa bacteria can be specifically detected. In addition, the ratio in the number of all oral microbial cells of A.a. microbe which became positive by this detection method was 0.005 to 0.032%.

(D) Detection of Bacteroides forsythus (Bf)
81, 95, 100, 202, 418, 423, 670, 700, 702, 704, 706, 708, 710 in the 16S rRNA gene sequence of Bf bacteria (Gene bank accetion number: X73962, SEQ ID NO: 4, FIG. 2) As the target bases that the 719, 721, 803, and 811th bases are recognized as cleavage points by the Cleavase enzyme (a restriction enzyme that specifically cleaves the 3 ′ side of the partial triplex structure), 18 first complements A pair of a strand and a second complementary strand was designed, and the detection sensitivity and specificity were examined using these. As a standard reagent for quantification, DNA was purified from Bf strain (ATCC43037), and the entire 16S rRNA gene sequence was PCR amplified and incorporated into a plasmid (pCRII-TOPO: Invitrogen), and this plasmid was used for detection sensitivity test. Plasmids were prepared in 10 3 to 10 7 7 copy / measurement tubes and used as standard reagents. In the detection sensitivity test, when the bases 81, 418, 423, 700, 704, 706, 708, 710, 721, and 803 are used as target bases in the above-described 20 sets of complementary strands of the invader assay method A measurement range of 10 3 to 10 7 copies / measurement tubes was obtained. Specificity tests were performed on these 10 sets of complementary strands. Saliva was selected as a sample for the specificity test. For DNA, 200 μl of saliva was extracted into 100 μl of eluate using MagNA Pure LC (Roche) and DNA Isolation kit I (Roche), treated at 95 ° C. for 5 minutes, and 5 μl was measured. used. Ten examples of DNA samples that became positive or negative by the real-time PCR method using the 563rd to 724th bases of the 16S rRNA gene of Bf bacteria as a gene amplification region were used for the above 10 sets of complementary strands. As a result of measurement by the invader assay method, a pair of complementary strands in which a difference from the result in the real-time PCR method was not recognized is a group having the 803th base (thymine) as a target base:

First complementary strand: 5′-CCCAGGTGGATTACTTAACGCTTTCGCTGTAGAGCTTACT-3 ′ (SEQ ID NO: 28)
Second complementary strand: 5′-CGCGCCGAGGACTATATCGCAAACTCCTAG-3 ′ (SEQ ID NO: 29)
(When this pair of complementary strands is used, a hybridization reaction is carried out by the invader assay method in the region of the underlined portion 5 of the 16S rRNA gene of Bf bacteria in FIG. 2, and the target base 51 is detected.)

  It was only. In addition, detection by invader assay using this pair of complementary strands was compared with quantitative analysis of 40 positive samples by real-time PCR method with real-time PCR method. A good correlation was obtained with a coefficient of 0.8013.

  In addition, detection by an invader assay method using a pair of complementary strands with the 803th base as a target base, Pg, Pi, Bf, Aa, Td, Neisseria subflava, Neisseria sicca, As a result of measuring 100 ng of DNA extracted from Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Streptococcus mitis, Streptococcus anguinosus, Streptococcus gordanii, Streptococcus sanguis strains, it was found that only Bf bacteria can be specifically detected. In addition, the ratio in the number of all oral microbial cells of B.f. microbe which became positive by this detection method was 0.011-1.815%.

(E) Detection of Treponema denticola (Td)
In the 16S rRNA gene sequence of Td bacteria (Gene bank accetion number: D85438, SEQ ID NO: 5, Fig. 2), the 151st, 155th, 420th, and 424th bases are selected from the Cleavase enzyme (specifically the 3 'side of the partial triplex structure). As a target base that is recognized as a cleavage point by the restriction enzyme that cleaves automatically, four pairs of first and second complementary strands are designed, and the detection sensitivity and specificity are examined using them. It was. As a standard reagent for quantification, DNA was purified from a Td strain (ATCC 35404), and the entire 16S rRNA gene sequence was PCR amplified and incorporated into a plasmid (pCRII-TOPO: Invitrogen), and this plasmid was used for detection sensitivity test. Plasmids were prepared in 10 3 to 10 7 7 copy / measurement tubes and used as standard reagents. In the detection sensitivity test, when the 155th and 420th bases are used as the target bases in the above-described 20 sets of complementary strands of the invader assay method, 10 3 to 10 7 copies / measurement tube A measurement range was obtained. Specificity tests were performed on these two sets of complementary strands. Saliva was selected as a sample for the specificity test. For DNA, 200 μl of saliva was extracted into 100 μl of eluate using MagNA Pure LC (Roche) and DNA Isolation kit I (Roche), treated at 95 ° C. for 5 minutes, and 5 μl was measured. used. Ten examples of DNA samples that became positive or negative by the real-time PCR method using the 333rd to 445th bases of the 16S rRNA gene of Td bacteria as the gene amplification region were used for the above two sets of complementary strands. As a result of measurement by the invader assay method, a pair of complementary strands in which a difference from the result in the real-time PCR method was not recognized is a group having the 155th and 420th bases (both as thymine) as the target base:
(When the 155th base is the target base)

First complementary strand: 5′-CGGAGCCGTAGCTCCTTTCCTCATTTACCTTTT-3 ′ (SEQ ID NO: 30)
Second complementary strand: 5′-CGCGCCGAGGATGTAAATGAGCACATTCG-3 ′ (SEQ ID NO: 31)
(When this pair of complementary strands is used, a hybridization reaction by the invader assay method is performed in the region of the underlined portion 6 of the 16S rRNA gene of Td bacteria in FIG. 2, and the target base 61 is detected.)

(When the 420th base is the target base)
First complementary strand: 5′-CATGACTACCGTCATCAAAGAAGCATTCCCTCTTCTTCTTT-3 ′ (SEQ ID NO: 32)
Second complementary strand: 5′-CGCGCCGAGGATTCTTCATCTGCAAAAGAATT-3 ′ (SEQ ID NO: 33)
(When this pair of complementary strands is used, a hybridization reaction is carried out by the invader assay method in the region of the underlined portion 7 of the 16S rRNA gene of Td bacteria in FIG. 2, and the target base 71 is detected.)

  Both were. Furthermore, when the detection by the invader assay method using these two types of complementary strands was compared with the real-time PCR method in 64 positive samples by the real-time PCR method, In the pair of complementary strands where the 155th base was the target base, the coefficient was 0.4757 by power regression, and in the pair of complementary strands where the 420th base was the target base, it was 0.9573.

  In addition, detection by invader assay using a pair of complementary strands with the 155th and 420th bases as target bases, Pg, Pi, Bf, Aa, Td, Neisseria subflava separated and cultured on a culture plate, As a result of measuring 100 ng of DNA extracted from strains of Neisseria sicca, Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Streptococcus mitis, Streptococcus anguinosus, Streptococcus gordanii, Streptococcus sanguis, it was possible to specifically detect only Td bacteria. The ratio of Td bacteria that became positive in this detection method to the total number of oral cells is 0.005 to 0.450% when the 155th base is the target base, and the 420th base is the target base. When it was, it was 0.007 to 0.507%.

It is drawing which showed the outline of the invader assay method. It is drawing which showed the positional relationship of the base sequence of 16S rRNA gene of a periodontal disease microbe, and the detection object area | region used in the Example of this invention.

Claims (11)

The following nucleotide chains (1) to (3):
(1) a template nucleotide chain having a target base to be detected;
(2) a first complementary strand having a base adjacent to the target base of the template nucleotide chain and capable of hybridizing from the adjacent base toward the 3 ′ side of the template nucleotide chain;
(3) having a base complementary to the target base, capable of hybridizing from the complementary base toward the 5 ′ side of the template nucleotide chain, and unable to hybridize toward the 3 ′ side, Two complementary strands;
To form a partial triplex structure in the target base portion of the template nucleotide chain (1), and then the 3 ′ side of the second complementary strand (3) that has formed the triplex structure The target base of the template nucleotide chain (1) is detected by detecting a nucleotide chain on the non-hybridizing side of the second complementary chain (3) released by the enzyme reaction by acting a restriction enzyme that specifically cleaves. In the method of detecting,
Codes 1-5 types of 16S rRNA selected from periodontal pathogens of the group consisting of Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Actinobacillus actinomycetemcomitans (Aa), Bacteroides forsythus (Bf), and Treponema denticola (Td) As a template nucleotide chain (1), the following (a) to (e) in the nucleotide chain:
(A) the base at position 748 of the gene encoding Pg 16S rRNA (including both complementary base sequences);
(B) the 191st base of a gene (including complementary base sequences) encoding Pi 16S rRNA;
(C) the 842nd base of the gene encoding 16S rRNA of Aa (including both complementary base sequences);
(D) the 842nd base of a gene (including complementary base sequences) encoding Bf 16S rRNA;
(E) the 155th base or the 420th base of a gene (including complementary base sequences) encoding Td 16S rRNA;
A method for detecting periodontal pathogens, wherein quantitative detection of these periodontal pathogens is performed by detecting as a target base. The method for detecting periodontal disease bacteria according to claim 1, wherein the detection method has at least one characteristic among the following groups (a) to (e).
(A) The 748th base of the gene encoding Pg 16S rRNA is guanine.
(B) The 191st base of the gene encoding Pi 16S rRNA is guanine.
(C) The 842rd base of the gene encoding Aa 16S rRNA is guanine.
(D) The 842th base of the gene encoding 16S rRNA of Bf is thymine.
(E) The 155th base of the gene encoding Td 16S rRNA is thymine and the 420th base is thymine. In the above detection method, the detected nucleotide chain (A) on the unhybridized side of the second complementary strand (3) is detected by detecting the nucleotide strand (A) and the probe (B):
(B): A structure in which a single nucleotide chain is bent and hybridized with the 3 ′ end and the 5 ′ end as both ends, and a part of the nucleotide chain on the 3 ′ end side protrudes as a single strand. (1) The protruding portion on the 3 ′ end side can hybridize with the above-described free nucleotide chain, and is further adjacent to the 5′-most base of the protruding portion, and further one base on the 5 ′ side. Is a target base (B1), (2) a probe in which a fluorescent dye (B2) is labeled in the vicinity of the 5 ′ end, and a fluorescent quencher (B3) is labeled in the vicinity of the 3 ′ side of the fluorescent dye;
The base part complementary to the target base at the 3 ′ end of the nucleotide chain (A), with a part or all of the protruding part on the 3 ′ end side of the probe (B) and the nucleotide chain (A). (A1) and the target base (B1) are hybridized to form a partial triple chain structure comprising the complementary base (A1), the target base (B1) and the base near the 5 ′ end of the probe (B). The 5 ′ end of the probe (B) together with the fluorescent dye (B2) labeled on the end portion is bound together with the probe (B2) by the action of a restriction enzyme that specifically cleaves the 3 ′ side of the triplex structure. The detection of periodontal pathogens according to claim 1 or 2, wherein the fluorescence intensity of the fluorescent dye (B2) released from the quenching action of the fluorescence quenching substance (B3) is used as an index. Method. In the above detection method, the first complementary strand is a nucleotide chain (complementary both) consisting of a base sequence of at least 9 bases from the target base (not including the base) in the template nucleotide chain (1) toward the 3 ′ side. A nucleotide chain that is complementary to the target base of the template nucleotide chain (1), and the total number of bases of the first complementary chain is 10 to 10 It is 50 bases, The detection method of the periodontal disease bacteria in any one of Claims 1-3 characterized by the above-mentioned. 5. The method for detecting periodontal disease bacteria according to claim 4, wherein in the detection method, the base sequence of the first complementary strand has at least one characteristic selected from the following (a) to (e).
(A) The base sequence for detection of Pg using the 748th base of a gene (including complementary both base sequences) encoding Pg 16S rRNA as the target base is the base sequence represented by SEQ ID NO: 6 ( Including both complementary base sequences).
(B) A base sequence represented by SEQ ID NO: 7 in the case of detecting Pi using the 191st base of a gene (including complementary both base sequences) encoding Pi 16S rRNA as the target base ( Including both complementary base sequences).
(C) The base sequence for detection of Aa using the 842nd base of a gene (including complementary both base sequences) encoding Aa 16S rRNA as the target base is represented by the base sequence represented by SEQ ID NO: 8 ( Including both complementary base sequences).
(D) A base sequence represented by SEQ ID NO: 9 in the case of detecting Bf using the 842nd base of a gene (including complementary both base sequences) encoding Bf 16S rRNA as a target base ( Including both complementary base sequences).
(E) When the Td is detected using the 155th base or the 420th base of the gene encoding Td 16S rRNA (including complementary both base sequences) as the target base, the base sequence is the 155th base. Is a base sequence represented by SEQ ID NO: 10 (including complementary both base sequences), and the 420th base is a base sequence represented by SEQ ID NO: 11 (including complementary both base sequences). In the above detection method, the sequence of the portion to be hybridized with the template nucleotide chain (1) of the second complementary strand is at least 10 from the target base (including the base) in the template nucleotide chain (1) toward the 5 ′ side. A nucleotide chain complementary to a nucleotide chain comprising a base sequence of bases (including complementary both base sequences), and the total number of bases in the hybridizing portion of the second complementary chain is 10 to 30 bases The method for detecting periodontal disease bacteria according to any one of claims 1 to 5, wherein: The said detection method WHEREIN: The base sequence of the part hybridized with the template nucleotide chain | strand (1) of a 2nd complementary strand has at least 1 type of characteristic chosen from following (a)-(e). Method for detecting periodontal disease bacteria.
(A) The base sequence in the case of detecting Pg using the 748th base of a gene (including complementary both base sequences) encoding Pg 16S rRNA as the target base is the base sequence represented by SEQ ID NO: 12 ( Including both complementary base sequences).
(B) A base sequence represented by SEQ ID NO: 13 in the case of detecting Pi using the 191st base of a gene (including complementary both base sequences) encoding Pi 16S rRNA as the target base ( Including both complementary base sequences).
(C) SEQ ID NO: 14 represents the first complementary strand when Aa is detected using the 842nd base of a gene (including complementary both base sequences) encoding Aa 16S rRNA as the target base. It is a base sequence (including both complementary base sequences).
(D) The first complementary strand in the case of detecting Bf using the 842nd base of a gene (including complementary both base sequences) encoding Bf 16S rRNA as a target base is represented by SEQ ID NO: 15. It is a base sequence (including both complementary base sequences).
(E) The first complementary strand in the case of detecting Td using the 155th base or the 420th base of a gene (including complementary both base sequences) encoding Td 16S rRNA as the 155th base Is the base sequence represented by SEQ ID NO: 16 (including complementary both base sequences), and the 420th base is the base sequence represented by SEQ ID NO: 17 (including complementary both base sequences). is there. In the above detection method, the total number of cells in the specimen is determined by the following nucleotide chain (1) to (3):
(1) a template nucleotide chain having a target base to be detected;
(2) a first complementary strand having a base adjacent to the target base of the template nucleotide chain and capable of hybridizing from the adjacent base toward the 3 ′ side of the template nucleotide chain;
(3) having a base complementary to the target base, capable of hybridizing from the complementary base toward the 5 ′ side of the template nucleotide chain, and unable to hybridize toward the 3 ′ side, Two complementary strands;
To form a partial triplex structure in the target base portion of the template nucleotide chain (1), and then the 3 ′ side of the second complementary strand (3) that has formed the triplex structure The target base of the template nucleotide chain (1) is detected by detecting a nucleotide chain on the non-hybridizing side of the second complementary chain (3) released by the enzyme reaction by acting a restriction enzyme that specifically cleaves. When quantitative detection is performed by the detection method and the ratio of the number of periodontal pathogens in the total number of cells is calculated, the target base to be detected by the detection method is a gene encoding Aa 16S rRNA. 1382th, Pg 1384th, Pi 1350th, Bf 1341st, and Td 1353th base (including complementary bases alternatively), and this 8. The method according to claim 1, wherein the method is performed by selecting a first complementary strand and a second complementary strand capable of commonly detecting target bases of different periodontal disease bacteria. A method for detecting periodontal disease bacteria according to claim 1. In the said detection method, the sample which provides a template nucleotide chain | strand (1) is a saliva sample or a plaque sample, The detection method of the periodontal disease bacteria in any one of Claims 1-8 characterized by the above-mentioned. A kit for performing the method for detecting periodontal disease bacteria according to claim 1, which has the following feature (1) and includes the first complementary strand and the second strand shown in the following (2) and (3): A kit for detecting periodontal disease bacteria, comprising a complementary strand of
(1) The target base is the following base.
(A) the 748th guanine of the gene encoding Pg 16S rRNA;
(B) the 191st guanine of the gene encoding Pi 16S rRNA;
(C) the 842rd guanine of the gene encoding Aa 16S rRNA;
(D) the 842 th thymine of the gene encoding Bf 16S rRNA;
(E) 155 th thymine or 420 th thymine of a gene encoding Td 16S rRNA;
(2) The first complementary strand is complementary to a nucleotide strand comprising a base sequence of at least 9 bases from the target base (not including the base) to the 3 ′ side. It has a nucleotide chain continuously with a base adjacent to the target base, and the total number of bases of the first complementary chain is 10 to 50 bases.
(3) For a nucleotide chain in which the sequence of the portion of the second complementary strand that is hybridized with the template nucleotide strand consists of a base sequence of at least 10 bases from the target base (including the base) toward the 5 ′ side And the total number of bases in the hybridizing portion of the second complementary strand is 10 to 30 bases. In the kit, for the purpose of quantitative detection of the total number of cells in the specimen, the target bases are the 1382th of the gene encoding 16S rRNA of Aa, the 1384th of Pg, and the same 1350 of Pi. No., Bf No. 1341 and Td No. 1353 base (complementary bases are included alternatively), and these different periodontal disease target bases can be detected in common The kit for detecting periodontal disease bacteria according to claim 10, comprising a first complementary strand and a second complementary strand.