JP2016214101A - Simple and quick inspection method of oral bacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and a tool which detect bacteria causing dental caries or the like simply and inexpensively at a dental clinic place.SOLUTION: There is provided a method for detecting oral bacteria including: 1) a process of amplifying nucleic acid in a sample by using two types or more of primer pairs corresponding to two or more oral bacteria, each of primer pairs being made up of a first primer for introducing a tag sequence associated with each oral bacteria and a second primer for introducing labeling substance binding substance; 2) a process of developing an amplification product on a solid-phase carrier containing two types or more of probes which include a sequence complementary with the tag sequence, by using developing liquid containing a labeling substance which enables detection through reaction with the labeling substance binding substance; and 3) a process of detecting a hybridized product of the amplification product and the probe, which is created on the solid-phase carrier. Also provided is a kit for the detecting method.SELECTED DRAWING: None

Description

  The present invention relates to a test method and test kit for oral bacteria using nucleic acid chromatography. More specifically, the present invention relates to a test method and test kit for oral bacteria that enables a semi-quantitative analysis of a plurality of bacteria by using a nucleic acid chromatography method.

  In the field of dentistry, identification of oral bacteria associated with periodontal disease and caries is important for elucidation of the etiology and treatment. Conventionally, for identification of oral bacteria, a method has been employed in which pathogenic bacteria in a specimen are amplified by PCR, and the amplified product is developed on an agarose gel by electrophoresis (Non-Patent Documents 1 and 2, etc.). . This method requires time and labor since the step of developing the amplification product in the PCR tube by agarose gel electrophoresis is indispensable. There is also a problem that specificity and sensitivity are not sufficient.

  A method for detecting bacteria more easily by using a DNA biosensor has been developed. In this method, a probe specific to a target bacterium is immobilized on the surface of the sensor, and the bacterium is detected by detecting luminescence generated by binding to bacterial DNA in the specimen. For example, a method for detecting bacteria using a visually detectable dipstick type sensor using colored polystyrene beads has also been reported (Non-Patent Document 3), but these methods detect 1-2 bacterial species. Yes, the subject is not oral bacteria. Furthermore, quantitative detection methods using quantitative RT-PCR, DNA microarrays, and surface plasmon resonance have been developed.

  On the other hand, Kawase et al. Have developed a simple and rapid multi-gene detection method called STH-PAS (Single Tag Hybridization-Printed Array Strip) (Patent Document 1). In this method, PCR amplification is performed by binding a single-stranded DNA tag to the PCR primer of the target bacterial nucleic acid, and the amplified product is developed on a membrane strip (PAS) printed on the line with oligo DNA complementary to the tag. A plurality of bacteria can be detected at the same time by trap-detecting the PCR amplification product by a hybridization reaction between single-stranded tag DNAs. If this STH-PAS can be applied to the detection of oral bacteria, it becomes possible to easily and inexpensively detect causative bacteria such as caries at the dentistry site.

WO2013 / 039228

Sato T. et al., Lett Appl Microbiol. 2003; 37 (1): 66-69 Mayanagi G. et al., Oral Microbiol Immunol. 2004 Dec; 19 (6): 379-385 D. P. Kalogianni, et al., Biosensors and Bioelectronics, 2009; vol. 24, no. 6, pp. 1811-1815

  An object of the present invention is to provide a method for detecting causative bacteria such as caries at a dental clinical site in a simple and inexpensive manner.

  The inventors made use of STH-PAS, which is a multigene detection method, to produce oral bacteria detection PAS (Printed Array Strip) printed with oligo DNA corresponding to five types of oral bacteria related to caries, It was confirmed that the presence or absence of the oral bacteria in the sample could be detected semi-quantitatively by directly expanding the amplification product in the PCR tube onto this strip.

That is, the present invention relates to the following [1] to [8].
[1] A method for detecting oral bacteria,
1) Two or more kinds of primer pairs corresponding to two or more oral bacteria, a first primer for introducing a tag sequence associated with each oral bacteria, and a first primer for introducing a labeling substance binding substance A step of amplifying a nucleic acid in a specimen using a primer pair comprising two primers;
2) On a solid phase carrier containing two or more kinds of probes containing sequences complementary to the tag sequence, using a developing solution containing a labeling substance that enables detection by reacting the amplified product with the labeling substance binding substance And 3) detecting the hybridization product of the amplification product and the probe produced on the solid phase carrier;
[2] The first primer includes the tag sequence and a first identification sequence complementary to the first base sequence of the oral bacterium, and between the tag sequence and the first identification sequence, The method according to [1] above, which has a ligation site capable of suppressing or terminating the DNA polymerase reaction, and wherein the second primer includes a second identification sequence complementary to the second base sequence of the oral bacterium. ;
[3] The connection site according to [1] or [2], wherein the linking site includes a C2-C40 alkylene chain or a polyoxyalkylene chain that is adjacent to a nucleotide in the primer via a phosphodiester bond. Method;
[4] Oral bacteria are Actinomyces oris, Mogibacterium timidum, Enterococcus faecalis, Porphyromonas endodontalis, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum, mutatum, Streptococcus The method according to any one of the above [1] to [3], which is any two or more selected from uli, Pseudoramibacter alactolyticus, Parvimonas micra, Treponema denticola, Eubacterium saphenum, Slackia exigua;
[5] Labeling substance binding substance and labeling substance are biotin and labeled avidin (labeled avidin), digoxigenin and labeled anti-digoxigenin antibody (labeled anti-digoxigenin antibody), or FITC and labeled anti-FITC antibody (labeled) The anti-FITC antibody), the method according to any one of [1] to [4] above;
[6] A kit for detecting oral bacteria,
1) Two or more types of primer pairs corresponding to two or more oral bacteria, each including a tag sequence associated with each oral bacteria and a first identification sequence complementary to the first base sequence of the oral bacteria A primer pair consisting of a first primer and a second primer for introducing a labeling substance binding substance,
2) a solid phase carrier containing two or more kinds of probes containing a sequence complementary to the tag sequence, and 3) a labeling substance that reacts with the labeling substance-binding substance and enables detection, or a developing solution containing the labeling substance,
A kit comprising:
[7] The oral bacteria are Actinomyces oris, Mogibacterium timidum, Enterococcus faecalis, Porphyromonas endodontalis, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Tanellaella forsythia, cusocria cus, eptococcus faecalis The kit according to [6] above, which is any two or more selected from Olsenella uli, Pseudoramibacter alactolyticus, Parvimonas micra, Treponema denticola, Eubacterium saphenum, Slackia exigua;
[8] Labeling substance binding substance and labeling substance are biotin and labeled avidin (labeled avidin), digoxigenin and labeled anti-digoxigenin antibody (labeled anti-digoxigenin antibody), or FITC and labeled anti-FITC antibody (labeled) Anti-FITC antibody), the kit according to [6] or [7] above.

  According to the present invention, it is possible to visually detect the presence of a plurality of oral bacteria without requiring a complicated procedure such as agarose gel electrophoresis. The method of the present invention does not require any special equipment or technology other than a simple desktop thermal cycler, and can easily and rapidly detect causative bacteria such as periodontal disease and caries at the dental medical site. Moreover, unlike the method using the conventional agarose gel electrophoresis, the method of the present invention can simultaneously detect a plurality of bacteria including not only pathogenic bacteria but also non-pathogenic bacteria semi-quantitatively.

FIG. 1 is a schematic diagram showing a strip used in the method of the present invention. Tag represents an oligonucleotide chain corresponding to the target bacterial DNA, and cTag represents a complementary chain thereof. FIG. 2 shows the result of developing the amplification product derived from the specimen on the strip. 1: Negative control, 2-6: Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces oris, Veillonella parvula in this order. FIG. 3 shows the result of verifying the detection limit by STH-PAS. 1-5: 10 ng, 1 ng, 100 pg, 10 pg, 1 pg in order. FIG. 4 shows the result of verifying the specificity of detection by STH-PAS. 1: Amplification product labeled with a single-stranded DNA tag at the 5 ′ end and labeled with biotin at the 3 ′ end, amplification product labeled with a single-stranded DNA tag at the 2: 5 ′ end, and biotin at the 3: 3 ′ end Target amplification product labeled with 4: Amplification product without label. FIG. 5 shows the results of semi-quantitative analysis by STH-PAS. 1-5: No dilution, 10 times, 10 ² times, 10 ³ times, 10 ⁴ times in order.

1. TECHNICAL FIELD The present invention relates to a method capable of simultaneously detecting two or more oral bacteria semi-quantitatively. The method for detecting oral bacteria of the present invention comprises:
1) Two or more kinds of primer pairs corresponding to two or more oral bacteria, a first primer for introducing a tag sequence associated with each oral bacteria, and a first primer for introducing a labeling substance binding substance A step of amplifying a nucleic acid in a specimen using a primer pair comprising two primers;
2) On a solid phase carrier containing two or more kinds of probes containing sequences complementary to the tag sequence, using a developing solution containing a labeling substance that enables detection by reacting the amplified product with the labeling substance binding substance And 3) detecting the hybridization product of the amplification product and the probe generated on the solid phase carrier.

1.1 Amplification Step In the present invention, the target oral bacterial nucleic acid is first amplified using a primer pair specific to the nucleic acid. In this amplification step, the tag sequence associated with each oral bacterium by the first primer is introduced into the nucleic acid of the target oral bacterium, and the second primer reacts (bonds) with a labeling substance to be described later for detection. Introduce a labeling substance binding substance that enables it.

(1) Oral Bacteria “Oral bacteria” that are detection targets of the present invention refer to pathogenic bacteria and non-pathogenic bacteria that are present in the oral cavity, particularly in the human oral cavity, and cause periodontal disease and caries. Specifically, for example, Actinomyces oris, Mogibacterium timidum, Enterococcus faecalis, Porphyromonas endodontalis, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Tannerella forsythia, Fusococcus nucleatum, ept Olsenella profusa, Olsenella uli, Pseudoramibacter alactolyticus, Parvimonas micra, Treponema denticola, Eubacterium saphenum, Slackia exigua (and above, non-pathogenic bacteria) and the like. In the present invention, two or more types of such oral bacteria can be detected simultaneously.

(2) Specimen The specimen used in the present invention is a sample containing nucleic acid (DNA or RNA) of the above oral bacteria, for example, oral plaque (plaque), biofilm, gingival crevicular fluid, tongue coating, Saliva, infected dentin, etc. are collected and nucleic acid extracted according to a conventional method can be used as a specimen.

(3) Primer The primer pair used in the present invention is two or more kinds of primer pairs corresponding to two or more oral bacteria, and the “first” for introducing a tag sequence associated with each oral bacteria. It consists of a primer pair consisting of a “primer” and a “second primer” for introducing a labeling substance binding substance.

  The “tag sequence” is a sequence designed so as to eliminate cross reaction between tags irrespective of the sequence of the target oral bacterial nucleic acid, preferably 15 to 50 bases, more preferably 15 It is not less than 25 bases. The oligonucleotide probe is designed to contain a sequence that is complementary to this tag sequence.

  The first primer and the second primer are respectively complementary to the first base sequence and the second base sequence in the oral bacterial nucleic acid so that they can hybridize to the oral bacterial nucleic acid. It has a “second identification sequence”. The first base sequence and the second base sequence are appropriately selected so that they can specifically hybridize and amplify with the oral bacterial nucleic acid. The DNA sequence of oral bacteria used in the present invention is already known, and the first base sequence and the second base sequence are set according to a conventional method based on the sequences.

  The terms “same” or “complementary” used in the present invention do not mean that they are completely identical or completely complementary. That is, the same as a certain sequence may include a sequence complementary to a base sequence that can be annealed to a certain sequence. On the other hand, complementary means a sequence that can anneal under stringent conditions.

  In the “first primer”, it is preferable that the tag sequence and the first identification sequence are not directly linked but have a linking site capable of suppressing or stopping the DNA polymerase reaction therebetween. This linking site does not contain a natural base or a derivative of a natural base (such as a natural base) that pairs with a natural base, and is composed of, for example, an artificial oligonucleotide, and thus suppresses or stops the DNA polymerase reaction. As a result, a nucleic acid having a single-stranded region is amplified, and hybridization with the probe becomes possible without thermal denaturation. For details of the introduction of the tag sequence and the linking site, refer to WO2013 / 039228.

  Examples of the linking site include a C2-C40 alkylene chain or polyoxyalkylene chain that is adjacent to the nucleotide in the primer via a phosphodiester bond.

More specifically, examples of the connecting site include those represented by any of the following formulas.
5′-O—C _m H _2m —O-3 ′ Formula (1)
(In the formula, 5 ′ represents an oxygen atom of a phosphodiester bond on the 5 ′ side, 3 ′ represents a phosphate atom of a phosphodiester bond on the 3 ′ side, and m represents an integer of 2 to 40. Represent),
Or
5 ′-(OC _n H _2n ) _l —O-3 ′ Formula (2)
(In the formula, 5 ′ represents an oxygen atom of a phosphodiester bond on the 5 ′ side, 3 ′ represents a phosphate atom of a phosphodiester bond on the 3 ′ side, and n represents an integer of 2 or more and 4 or less. And l is an integer of 2 or more, and (n + 1) × l represents an integer of 40 or less.)

  The “second primer” includes, in addition to the second identification sequence, a labeling substance binding substance that reacts with the labeling substance and allows visual confirmation when the amplified oral bacterial nucleic acid hybridizes with the probe.

(4) Nucleic acid amplification The nucleic acid amplification method is not particularly limited, and any method such as a PCR method, an SDA method, a LAMP method, an ICAN method, or an RCA method may be used, but typically the PCR method is used. Do it. By performing amplification using the above-described primer pair, a tag sequence is introduced into one end of the double-stranded nucleic acid that is an amplification product, and a labeling substance binding substance is introduced into the other end.

1.2 Development / Hybridization Step Next, the amplification product is developed on a solid phase carrier on which two or more probes corresponding to two or more oral bacteria are immobilized. The specimen containing the amplification product is easily developed by the capillary phenomenon in the porous material constituting the solid phase carrier.

(1) Developing solution “Developing solution” is added to the amplification reaction solution containing the amplification product so as to facilitate the development of the nucleic acid amplification product in the solid phase carrier. The development medium is not particularly limited, and examples thereof include water, an organic solvent compatible with water, or a mixture of water and one or more organic solvents. Examples of the organic solvent compatible with water include lower alcohols having about 1 to 4 carbon atoms, esters such as DMSO, DMF, methyl acetate, and ethyl acetate, acetone, and the like. The development medium is preferably mainly water.

  The development medium can include a buffer component for adjusting the pH. The buffer component is usually in the range of 6.0 to 8.0, depending on the intended pH. More preferably, it is 7.0 or more and 8.0 or less. Components for obtaining such pH are, for example, acetic acid and sodium acetate (acetic acid buffer), citric acid and sodium citrate (citrate buffer), phosphoric acid and sodium phosphate (phosphate buffer), and the like. Furthermore, a phosphate buffered saline (PBS) etc. are mentioned. In addition, you may employ | adopt an amplification reaction liquid as a developing medium as it is. Further, the composition and concentration may be adjusted by adding an additional component such as a surfactant or an appropriate salt or a solvent to the amplification reaction solution, and the resultant may be used as a developing medium. The development time is not particularly limited, and is appropriately set according to the form and shape of the solid support and the properties of the development medium.

  A labeling substance is added to the developing solution. By doing so, a double-stranded nucleic acid having a labeling substance as a complex product bound to the labeling substance-binding substance introduced into the double-stranded nucleic acid that is the amplification product is obtained.

  In addition to the above method, by carrying out an amplification reaction by adding a labeling substance in advance in the amplification step, the labeling substance has a labeling substance as a complex product bound to the labeling substance binding substance after the amplification step. A double-stranded nucleic acid may be obtained. In addition, a complex product may be obtained in the same manner by adding a labeling substance to the double-stranded nucleic acid in the amplification reaction solution after completion of the reaction. Such modifications are also included in the scope of the present invention.

(2) Solid-phase carrier The “solid-phase carrier” constituting the device of the present invention has a porous material that holds a probe that can hybridize with a probe associated with an oral bacterium to be detected. The solid phase carrier may be composed of a porous material, or may be composed of a porous material (porous sheet) and a backing member that supports the porous material. The porous material constituting the solid phase carrier is well known in the art, and examples thereof include cellulose, nitrocellulose, and nylon. The backing member is not particularly limited as long as it has a property of not allowing water to pass through (not evaporating), and a material known in the art can be used.

The shape of the solid phase carrier is appropriately designed according to the device so as to be suitable for the purpose of the present invention. For example, when the device is composed of a solid support coated with a transparent film and the detection is performed in a microtube having a volume of about 1.5 μl, the end of the solid support is immersed in the test solution in the tube. Designed to size and shape. In addition, when the test solution is applied to the solid phase carrier via a sample port or the like, each oral bacterial nucleic acid is designed to be identifiable in size and shape after the test solution is developed. The size of the solid phase carrier is typically, but not limited to, a flat area of 150 mm ² or less, an aspect ratio of 1.5 to 20 and a thickness of 0.01 mm to 0.3 mm. Can do.

  The test solution (developing solution) is applied to the end of the solid phase carrier, and the probe is fixed in parallel to each corresponding oral bacteria at a position away from the end by a certain amount.

  A position marker may be arranged on the solid phase carrier so that a probe region corresponding to each oral bacteria (tag sequence associated with) can be easily identified even when a plurality of oral bacteria are detected simultaneously. Good. Due to the presence of the position marker, even in the case of visual detection, it is possible to easily detect the presence or absence of the target oral bacteria.

(3) Probe The “probe” is designed to have a sequence complementary to the tag sequence. The length of the probe is not particularly limited, but is preferably 15 bases or more and 50 bases or less in order to ensure specificity for each tag sequence and hybridization efficiency. More preferably, it is 15 bases or more and 25 bases or less.

  The method for immobilizing the probe is not particularly limited, and may be bound to the solid phase carrier at the 3 'end or may be bound at the 5' end. For example, when nitrocellulose or the like is used for a solid phase carrier, the sticking property is enhanced by UV irradiation or the like after the probe is printed on the solid phase carrier by an ink jet method. In this method, the probe is immobilized only on the surface of the solid support where UV reaches.

  Since the “probe” contains a sequence complementary to the tag sequence associated with each oral bacterium, if the sample contains the nucleic acid of the target oral bacterium, the amplified product will hybridize with the corresponding probe and its position. To be captured.

(4) Labeling substance The “labeling substance” is preferably a luminescent substance or a coloring substance that presents luminescence or color development that can be detected visually (with the naked eye). Such labeling substances include chemiluminescent substances including various dyes, various pigments, luminol, isoluminol, acridinium compounds, olefins, enol ethers, enamines, aryl vinyl ethers, dioxene, arylimidazoles, lucigenin, luciferin and eclion. It is done. Furthermore, colloids or sols including gold colloids or sols or silver colloids or sols, metal particles, inorganic particles and the like can be mentioned.

  A part of the labeling substance may have particles such as latex particles. The average particle diameter of particles such as latex particles constituting a part of the labeling substance is not particularly limited, but is, for example, 20 nm to 20 μm, typically 40 nm to 10 μm, preferably 0.1 μm to 10 μm, particularly Preferably they are 0.1 micrometer or more and 5 micrometers or less, More preferably, they are 0.15 micrometers or more and 2 micrometers or less. Moreover, it adjusts suitably according to the hole diameter of a solid-phase carrier.

  Preferably, the particles are particles that can be suspended in an aqueous solution and consist of a water-insoluble polymeric material. Examples include polyethylene, polystyrene, styrene-styrene sulfonate copolymer, acrylic acid polymer, methacrylic acid polymer, acrylonitrile polymer, acrylonitrile-butadiene-styrene, polyvinyl acetate-acrylate, polyvinyl pyrrolidone, or vinyl chloride-acrylate. Mention may also be made of latex particles having active groups on their surface, for example carboxyl, amino or aldehyde groups.

  Representative examples of the labeling substance binding substance include antibodies in antigen-antibody reactions, biotin in an avidin (streptavidin) -biotin system, digoxigenin in an anti-digoxigenin (DIG) -digoxigenin (DIG) system, or FITC in an anti-FITC-FITC system. Haptens and the like. The labeling substance used for detection is bound to the other molecule or substance that interacts with the labeling substance-binding substance (for example, an antigen, ie, streptavidin, anti-FITC, etc.), and in the hybridization step with the probe, Alternatively, prior to or after this step, the amplified product is detected by binding to the labeling substance binding substance of the amplification product and a labeling substance having a site that binds to the labeling substance binding substance.

1.3 Hybridization / detection Amplified products derived from oral bacteria are captured by probes having a sequence complementary to the tag sequence associated with each oral bacteria while being developed in the solid phase carrier, and light emitted by the labeling substance. Or color development occurs. Thus, a plurality of nucleic acids can be visually detected at a time by light emission or color development of a line in which a probe corresponding to a tag bonded to each oral bacterial nucleic acid is immobilized.

  Since luminescence or color development from the labeling substance depends on the amount of nucleic acid derived from oral bacteria, oral bacteria can be detected semi-quantitatively by the amount (intensity) of luminescence or color development.

  After hybridization, a washing step may be further included as appropriate. When the amplified nucleic acid subjected to the hybridization step has a labeling substance, a special labeling step is not necessary. When the amplified nucleic acid binds to the labeling substance, a step of reacting with the labeling substance is required further or prior to the hybridization.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

1. Materials and Methods (1) Bacterial strains Five bacterial strains, Streptococcus mutans NCTC10449, Streptococcus sobrinus 6715, Scardovia wiggsiae F0424, Actinomyces oris WVU627 and Veillonella parvula ATCC10790, CDC anaerobic 5% sheep blood agar (BD, Flanklin Lake) Incubate on plate and in anaerobic glove box (model AZ-Hard, Hirasawa, Tokyo, Japan) for 3 days at 37 ° C under 80% N ₂ , 10% H ₂ and 10% CO ₂ gas phase .

(2) Collection of subjects and samples Enamel without caries with informed consent from 16 healthy subjects (average age, 31.8 ± 8.9 years; range, 23-54 years) Plaques (1.5 mg) on the gingival margin were sampled from the textured surface. Caries status was recorded using the DMFT index (untreated caries, lost teeth and teeth treated due to caries). The third molar was excluded. The subject has not taken antibiotics for 3 months prior to sampling. Plaque samples were collected using a sterile toothpick and stored at −20 ° C. in a 1.5 mL sampling tube until analysis.

(3) DNA extraction Genomic DNA was extracted from plaque or 3-day bacterial culture using InstaGene Matrix kit (Bio-Rad Laboratories, Richmond, CA) according to product instructions.

(4) PCR amplification Direct PCR was performed using the tagged species-specific primers listed in Table 1. Each PCR mix contains 21 μL plaque sample DNA or bacterial genomic DNA, 25 μL Taq DNA polymerase (HotStar Taq PLUS Master Mix; Qiagen GmbH, Hilden, Germany) and 4 μL primer mix. PCR amplification, 30 cycles of 95 ° C, 5 min initial heat activation, 95 ° C, 1 min denaturation, appropriate annealing temperature (see Table 1), 1 min annealing and 72 ° C, 1.5 min extension Then, it was set to 72 ° C. and extension for 10 minutes, and performed using PCR Thermal Cycler MP (TaKaRa Biomedicals, Ohtsu, Shiga, Japan) or iCycler (Bio-Rad Laboratories).
Actinomyces species include A.oris, A.naeslundii, and A.odontolyticus.
“X” represents spacer C3.
The sequence of each primer is shown in SEQ ID NOs: 1 to 10 in the sequence listing from the top.

(5) Dipstick DNA chromatography Commercially available dipstick DNA strips and reagents were used (TBA Co., Sendai, Japan). As shown in Table 1, for each pair of primers, the 5 ′ end (of the forward primer) was tagged with 5 different oligonucleotides and the 5 ′ end (of the reverse primer) was biotinylated. A spacer C3 was inserted between each tag and the forward primer so that DNA synthesis was terminated at this insertion site. Incidentally, the spacer C3 is (represented in FIG. 1 (b) and in Table 1 "X") spacer made of three carbon, (CH _{2) 3} [phosphoramide C3 spacer (Glen Research, Sterling, VA)] were used .

Blue latex particles coated with streptavidin were bound to the amplified product with biotinylated ends by streptavidin-biotin interaction. Five tag-complementary oligonucleotides were immobilized on dipstick strips (2.5 mm x 45 mm) in order to specifically capture PCR amplifications by hybridization with 5 'end tags. Of the total 12 available test lines on the strip, the first 5 were selected for this study. A biotin-fixed flow control line was set at the end of the strip (FIG. 1). One PCR amplification product is diluted to a total volume of 30 μL, 10 μL of diluent (containing detergent, blocking agent, PBS and salt solution) and 2 μL of streptavidin-coated blue latex suspension (both of which are Supplied by TBA Co.). A DNA strip was then inserted into the mixture. As a result, when the target DNA is captured by the tag-complementary oligonucleotide, the presence of the target DNA sequence can be visually confirmed by a blue test line (width 0.5 mm; mutually spaced 1.2 mm).

(6) Sensitivity of dipstick DNA chromatography To confirm the sensitivity of dipstick DNA chromatography, aliquots containing 10 ng, 1 ng, 100 pg, 10 pg or 1 pg of DNA amplified by PCR from 5 bacterial strains (aliquots) ) Was prepared and analyzed simultaneously. The detection limit of dipstick DNA chromatography for each bacterial strain was determined by visual inspection.

(7) Specificity and sensitivity of PCR dipstick DNA chromatography
The specificity of PCR dipstick DNA chromatography was confirmed under the following conditions: (i) a primer without oligonucleotide; (ii) a primer without biotin; and (iii) no target DNA template.

  The cross-reaction between the primer and other bacteria was previously tested by in vitro experiments using typical oral bacteria. Furthermore, in order to confirm the specificity of the primer in silico, a BLAST search was performed. S. sobrinus primers cross-reacted with Streptococcus downei, and Actinomyces species (Actinomyces oris, Actinomyces naeslundii and Actinomyces odontolyticus) were found to cross-react with Micromonospora peucetia, but S. downei and M. peucetia were found in the human oral cavity. There are no reports of separation.

  To assess the sensitivity of the assay, PCR was amplified from serial 10-fold dilutions of DNA purified from each bacterial strain (range: 10 ng to 1 fg) and divided into two (equal volumes). One was used for dipstick DNA chromatography to determine the detection limit. The other was analyzed by 1% agarose gel electrophoresis (high intensity analytical grade agarose, Bio-Rad Laboratories) as follows. PCR products (10 μL each) were separated on a 1% agarose gel in Tris-borate EDTA buffer (100 mM Tris, 90 mM borate, 1 mM EDTA, pH 8.4), stained with ethidium bromide, UV Taken below. A 100 bp DNA ladder (Invitrogen Corp., Carlsbad, Calif.) Was used as a molecular size marker.

(8) Semi-quantitative assay for plaque samples Semi-quantitative analysis of clinical samples requires accurate optimization and standardization. DNA was extracted from plaque samples and amplified by PCR for each bacterial species (Table 2). The PCR amplification product was diluted 10-fold from the original concentration to ¹⁰⁴ times. 1 μL aliquots with the same dilution factor were mixed for each bacterial species and detected by dipstick DNA chromatography. The detection limit of each bacterial species in dental plaque is obtained from a continuous chromatogram and the relative concentration of each bacterial species is corrected by using the relative sensitivity of PCR dipstick DNA chromatography as described above. The detection limit was estimated based on the dilution factor.

2. Results and Discussion (1) Validation of Dipstick DNA Chromatography Principle In the reaction mixture, biotin-labeled PCR amplifications immediately bind to streptavidin-coated blue latex particles due to the strong biotin-streptavidin affinity, A hybrid was formed. When the tip of the dipstick strip is inserted into the mixture, the PCR amplification-blue latex hybrid is moved up through the strip by capillary action and is anchored as a test line on the strip. And accumulated blue latex microspheres, resulting in a visible blue test line. A flow control line at the other end of the strip set the assay to function properly so that the total amount of latex particles was able to capture total DNA amplification, as well as excess latex particles. Various DNA amplifications could be detected within 5 minutes alone, or after 5-10 minutes in one assay simultaneously without cross-reaction (Figure 2). From the above, it was confirmed that the method of the present invention is suitable for simultaneous analysis of a plurality of bacteria.

  Development of dipstick biosensors is entering the stage of analyzing multiple objects simultaneously. A method of simultaneously detecting a plurality of alleles using a biosensor having two or more test lines has also been proposed. These biosensors usually require further steps in the assay, namely primer extension (PEXT) or oligonucleotide ligation reaction (OLR). The method of detecting dsDNA (double stranded DNA) maintains the simplicity of a dipstick DNA biosensor. Such a DNA biosensor uses a reporter, a streptavidin-biotin system as a capture mechanism, and an oligonucleotide that binds to a PCR amplification product, but can detect only one target dsDNA amplification product. In contrast, in the method of the present invention, a plurality of dsDNA sequences can be simultaneously detected in one assay by immobilizing oligonucleotides complementary to five types of tags on one strip.

(2) Sensitivity of dipstick DNA chromatography The detection limit of dipstick DNA chromatography was evaluated using purified DNA amplified from each bacterial strain in order to exclude the influence of the PCR method on sensitivity (FIG. 3). ). For all five species, PCR products could be detected at a level of 100 pg. Intensity was observed in proportion to DNA concentration. When the total amount of DNA product was tested in a useful analytical range below saturating concentration, intensity provided quantitative information for the target DNA. From this, it was confirmed that semi-quantitative analysis was possible by the method of the present invention.

(3) Specificity and sensitivity of PCR dipstick DNA chromatography In this study, five test lines generated by the target DNA hybrid appeared in the expected location. When 5 target DNA amplifications were detected in one assay, no smeared or false positive lines were observed on the strip. The result of the specificity evaluation is shown in FIG. Analysis performed with primers without oligonucleotide or without biotin or in the absence of target DNA template did not yield a positive signal. Under these three conditions, streptavidin-coated blue latex particles were unable to bind to the oligonucleotide detector to produce a blue signal in the test zone and were eventually captured in the control line. . The specificity of dipstick DNA chromatography using the same biosensor has also been confirmed in another experimental system that has detected 176 bacterial strains. Furthermore, no cross-reaction with other microorganisms was observed in silico or in vitro (in this study), indicating the high specificity of the method of the invention.

  A comparison of detection sensitivities obtained using PCR dipstick DNA chromatography and PCR-agarose gel electrophoresis is shown in Table 3. Although there were differences in the five bacterial species, PCR dipstick DNA chromatography was at least 10 times more sensitive than agarose gel electrophoresis.

(4) Semi-quantitative determination of plaque samples From PCR dipstick DNA chromatography using purified bacterial DNA, the sensitivity of this method varies among bacterial species (Table 3), but the detection limits are the same for all five species. (FIG. 3). Therefore, the difference in detection sensitivity is considered to be derived from the efficiency of PCR amplification. S. mutans, S. sobrinus, S. wiggsiae, A. oris and V. The relative PCR amplification efficiencies (E _r ) of parvula were estimated to be 1, 10 ⁻⁴ , 10 ⁻³ , 10 ⁻³ and 10 ⁻² from Table 3, respectively. For this reason, the relative concentration of bacteria (C _r ) was calculated by multiplying by the dilution factor (D) for the detection limit, dividing by the relative PCR amplification efficiency (E _r ), and using the following equation:
C _r = D / E _r (1)
C _r is the relative concentration of a given bacterium in the plaque; D is the dilution factor at the limit of detection; E _r is the relative PCR amplification efficiency.

  A PCR dipstick DNA chromatography assay was applied to 16 gingival plaque samples. A chromatogram was acquired for each sample (Figure 5). Actinomyces species were detected in all 16 samples with consistently high DNA concentrations. S. mutans and V. parvula was present in 15 of 16 samples. Although frequently detected, the amounts of these two species differed significantly between samples. S. sobrinus is detected in only one sample with the lowest detection frequency. Wiggsiae was detected in 4 out of 16 samples. At least two of the five species tested were detected in plaque samples on the gingival margin.

Semi-quantitative analysis was performed based on the above calculations (Table 4). From the relative concentration of each bacterial species, the composition of the oral microflora could be detected easily and quickly. Take sample 1 as an example (FIG. 5). sobrinus and S. There was no wiggsiae. Among the three positive species detected, V. parvula, Actinomyces sp. It was dominant over mutans. S. mutans, Actinomyces species and V. The relative concentration of parvula was estimated to be 1:10 ⁵ : 10 ⁵ . Actinomyces species and V. parvula was observed as the predominant bacterium in all samples tested. S. Mutans showed a high detection frequency, but a lower proportion of plaque bacteria overall when compared to other previous reports. This may be due to the low detection limit of PCR dipstick DNA chromatography and the use of different primers (unlike this study). In subjects with low, moderate and high DMFT (Table 4), there was no significant difference in the detection frequency (and amount) of the five bacteria.

  From the above, it was confirmed that a plurality of oral bacteria can be easily and rapidly detected semi-quantitatively simultaneously by the method of the present invention.

  The present invention can semi-quantitatively detect oral bacteria without requiring any special equipment or technology other than a simple desktop thermal cycler. Therefore, it is useful as a method / tool that can easily and quickly detect causative bacteria such as periodontal disease and caries in a dental field.

Sequence number 1: Synthetic DNA (Sm1 primer)
Sequence number 2: Synthetic DNA (Sm2 primer)
Sequence number 3: Synthetic DNA (SobF primer)
Sequence number 4: Synthetic DNA (SobR primer)
Sequence number 5: Synthetic DNA (Scar448F primer)
Sequence number 6: Synthetic DNA (Scar619R primer)
Sequence number 7: Synthetic DNA (AcoF primer)
Sequence number 8: Synthetic DNA (AcoR primer)
Sequence number 9: Synthetic DNA (VP2696F primer)
Sequence number 10: Synthetic DNA (VP3318R primer)

Claims (8)

A method for detecting oral bacteria,
1) Two or more kinds of primer pairs corresponding to two or more oral bacteria, a first primer for introducing a tag sequence associated with each oral bacteria, and a first primer for introducing a labeling substance binding substance A step of amplifying a nucleic acid in a specimen using a primer pair comprising two primers;
2) On a solid phase carrier containing two or more kinds of probes containing sequences complementary to the tag sequence, using a developing solution containing a labeling substance that enables detection by reacting the amplified product with the labeling substance binding substance And 3) detecting a hybridization product of the amplification product and the probe generated on the solid phase carrier.   The first primer includes the tag sequence and a first identification sequence complementary to the first base sequence of the oral bacterium, and a DNA polymerase reaction is performed between the tag sequence and the first identification sequence. 2. The method according to claim 1, wherein the second primer includes a second identification sequence that has a linking site capable of suppressing or stopping the second primer, and wherein the second primer is complementary to the second base sequence of the oral bacterium.   3. The method of claim 1 or 2, wherein the linking site comprises a C2-C40 alkylene chain or polyoxyalkylene chain that is adjacent to a nucleotide in the primer via a phosphodiester bond.   Oral bacteria, Actinomyces oris, Mogibacterium timidum, Enterococcus faecalis, Porphyromonas endodontalis, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Veillonella parvula, Olsenella profusa, Olsenella uli, Pseudoramibacter The method according to any one of claims 1 to 3, which is any two or more selected from alactolyticus, Parvimonas micra, Treponema denticola, Eubacterium saphenum, and Slackia exigua.   The method according to any one of claims 1 to 4, wherein the labeling substance-binding substance and the labeling substance are biotin and labeled avidin, digoxigenin and labeled anti-digoxigenin antibody, or FITC and labeled anti-FITC antibody. A kit for detecting oral bacteria,
1) Two or more types of primer pairs corresponding to two or more oral bacteria, each including a tag sequence associated with each oral bacteria and a first identification sequence complementary to the first base sequence of the oral bacteria A primer pair consisting of a first primer and a second primer for introducing a labeling substance binding substance,
2) a solid phase carrier containing two or more kinds of probes containing a sequence complementary to the tag sequence, and 3) a labeling substance that reacts with the labeling substance-binding substance and enables detection, or a developing solution containing the labeling substance,
Including kit.   The oral bacteria are Actinomyces oris, Mogibacterium timidum, Enterococcus faecalis, Porphyromonas endodontalis, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum, Streptococcus The kit according to claim 6, which is any two or more selected from Pseudoramibacter alactolyticus, Parvimonas micra, Treponema denticola, Eubacterium saphenum, and Slackia exigua.   The kit according to claim 6 or 7, wherein the labeling substance-binding substance and the labeling substance are biotin and labeled avidin, digoxigenin and labeled anti-digoxigenin antibody, or FITC and labeled anti-FITC antibody.