Patent Description:
Periodontal disease and dental caries, the two major oral diseases, are bacterial infectious diseases involving multiple bacteria.

Streptococcus mutans, Streptococcus sobrinus and Lactobacillus are known as causative bacteria of dental caries. These causative bacteria are known to metabolize sugars to produce lactic acid, as a result of which oral environment becomes acidic and enamel decalcification occurs.

It has been reported that, in addition to the amount of existing causative bacteria, the ratio of Streptococcus mutans to the bacterial count of Streptococci existing in the oral cavity can be utilized as an index. Specifically, when the ratio of Streptococcus mutans to the bacterial count of Streptococci is high, dental caries is easily caused (for example, see Non-Patent Documents <NUM>-<NUM>).

For a bacterial examination for dental caries, a kit is commercially available which examines by culturing Streptococcus mutans and Lactobacillus, and is utilized as one of the materials for diagnosis. The resulting cultures are visually observed to measure approximate colony counts and examination results are assessed.

However, since culture conditions of Streptococcus mutans differ from those of Lactobacillus, these must be cultured separately, and there was no technique for measuring them efficiently and simultaneously. Moreover, according to such a culture method, several days are required for the measurement, and there is a problem that it takes time to obtain assessment results.

<NPL>, discusses design and validation of <NUM> rRNA probes to enumerate members of the Clostridium leptum subgroup in human faecal microbiota.

<NPL> discusses a DNA Microarray Platform Based on Direct Detection of rRNA for Characterization of Freshwater Sediment-Related Prokaryotic Communities.

<CIT> relates to simple methods of determining the risk of periodontitis and caries (dental caries) simultaneously.

Under such circumstances, it has been desired to develop, as a bacterial examination for dental caries, a means/method with which it is possible to quickly and efficiently detect causative bacteria of dental caries (a plurality of types of bacterial groups) and assess dental caries.

The present invention was made in consideration of the above-described circumstances.

The present invention is defined in the claims and provides a DNA chip, which carries probes (a) and at least one probe from among a probe (b) and a probe (c):.

wherein the types of the probes are distinguished by the nucleotide sequences.

The present invention also provides a probe set for detecting dental caries bacteria, which comprises probes (a) and at least one probe from among a probe (b) and a probe (c):.

Further aspects of the invention are also defined in the claims.

According to the present invention, as a bacterial examination for dental caries, it is possible to provide a means/method with which it is possible to quickly and efficiently detect causative bacteria of dental caries (a plurality of types of bacterial groups) and assess dental caries.

Specifically, according to the present invention, the respective bacterial counts of Lactobacilli, Streptococcus sobrinus and Streptococci can be simultaneously calculated in a short time by using a DNA chip that carries probes corresponding to DNA sequences specific to the respective bacteria, as defined in the claims.

The probe (a) and the probe (b) and/or the probe (c), as defined in the claims, can be used as a probe set for detecting dental caries bacteria.

Hereinafter, each of these probes will be explained.

An oral bacterium to be detected is preferably two or more of Lactobacilli, Streptococcus sobrinus and Streptococci, as defined in the claims.

The probe (a) can hybridize to a nucleotide sequence of a specific region in a nucleotide sequence of a nucleic acid derived from the oral bacterium. In this regard, the nucleic acid is preferably a chromosomal DNA.

Probes that can be used for the present invention are defined in the claims and designed from regions that can serve as specific nucleotide sequences for <NUM> rRNA gene in the chromosomal DNA of the above-described respective intraoral bacteria to be detected. In general, probe design requires, in addition to selection of specific regions, matching of melting temperatures (Tm) and minimization of formation of a secondary structure.

Meanwhile, the specificity of the probe may be such that it allows collective detection of bacteria belonging to the same genus based on the specificity at a genus level, or such that it allows detection based on the specificity at an individual species level, which can be suitably selected and determined according to the purpose of the bacteria detection. Regarding the length of the probe, for example, a sequence of <NUM> bases or longer, and preferably <NUM> bases or longer, and <NUM> bases or shorter, and preferably <NUM> bases or shorter can be employed.

Examples of the probe (a) are shown in Table <NUM> (SEQ ID NOS: <NUM>-<NUM>).

The probe to be used for the present invention can be prepared, for example, through chemical synthesis employing a general oligonucleotide synthesis method. Such a probe can be designed, for example, by Probe Quest (registered trademark: manufactured by Dynacom). At the time of design thereof, it is required to set stringent conditions in consideration of conditions at the time of hybridization.

The term "stringent conditions" as used herein refers to conditions that are less likely to cause cross-hybridization induced by similar sequences, or that can dissociate any nucleic acids cross-hybridized with similar sequences. Specifically, it refers to the conditions for washing the DNA chip upon or after the hybridization reaction. For example, it is preferred that the salt concentration of a buffer is <NUM> to <NUM>, and that the temperature thereof is <NUM> to <NUM>. As more stringent conditions, as conditions under which the probe sequences shown in Table <NUM> hybridize and other similar sequences do not hybridize, it is more preferred that the salt concentration is <NUM> to <NUM> and that the temperature is <NUM> to <NUM>. Specific examples of conditions include those of <NUM> and <NUM>.

Examples of DNA to be hybridized include a nucleotide sequence having at least <NUM>%, e.g., at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% homology (identity) to the nucleotide sequence of DNA of the probe, as defined in the claims.

The nucleotide sequence of the nucleic acid of the above-described intraoral bacterium to be detected in the present invention does not have to be the exact nucleotide sequence thereof, as defined in the claims, and for example, it may have partial mutations such as deletion, substitution and insertion in the nucleotide sequence.

Specific preferred examples of the probe to be used in the present invention include those consisting of a nucleotide sequence DNA of (i), (ii) or (iii) below:.

In this regard, the "sequence substantially identical" regarding the sequence of (iii) above is a nucleotide sequence at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% homology (identity) to the sequence represented by any of SEQ ID NOS: <NUM>-<NUM> or the complementary sequence thereof.

The total amount indicator probe is a probe intended to capture all of the bacteria in the specimen that were successfully amplified with specific primer pairs. With respect to bacteria detection, it is crucial to detect the total amount of bacteria from the viewpoint of how much the bacteria targeted for detection exist among the entire bacteria including bacteria not targeted for detection, and also from the viewpoint of how much level of bacteria are present in the specimen in the first place.

The bacteria not targeted for detection are understood as a sum (total) of bacteria whose presence and types are known but not targeted for detection, and bacteria whose presence and types are unknown.

For the detection of the total amount of bacteria, for example, the total amount of bacteria may be measured independently from the DNA chip, but convenience of handling/measurement would be enhanced by providing a probe that can serve as an index of the total amount of bacteria on the DNA chip. The probe to be used may be a nucleotide sequence that is common to various types of bacterial species among the nucleotide sequences amplified by the primer pairs. If no such sequence is found, a plurality of relatively common sequences may be designed to be subjected to comprehensive judgement, thereby providing a total amount indicator probe. The total amount indicator probe is preferably a probe that hybridizes to nucleic acids derived from the bacteria contained in the specimen, specifically, a probe that contains a nucleotide sequence commonly included by a plurality of types of bacteria targeted for detection among the nucleotide sequences amplified by the aforementioned specific primer pairs.

An example of the total amount indicator probe is shown in Table <NUM> (SEQ ID NO: <NUM>).

An absolute amount indicator probe refers to a probe that hybridizes only to the nucleic acids of the absolute amount indicator.

As used herein, the absolute amount indicator refers to nucleic acids that are added to the specimen in a certain amount prior to amplification reaction and hybridization reaction. The absolute amount indicator is nucleic acids that ensure actual amplification reaction upon usual amplification reaction, and plays a role as a so-called positive control.

Therefore, when the DNA chip is provided with a probe specific to the absolute amount indicator, it can be confirmed from detection results thereof whether or not amplification reaction, hybridization and the like have been appropriately performed. Moreover, if one type of absolute amount indicator is set, the fluorescence intensity of said absolute amount indicator acquired from multiple DNA chips should be constant, and thus the fluorescence intensities of the absolute amount indicator may be compared to calculate correction coefficients if there are some variations in the amplification efficiency or the hybridization efficiency. The corrected fluorescence intensities can be used upon comparison among the multiple DNA chips.

Examples of the absolute amount indicator probe are shown in Table <NUM> (SEQ ID NOS: <NUM>-<NUM>). Further, examples of nucleotide sequences which can serve as the absolute amount indicator (nucleotide sequences targeted for the detection using the absolute amount indicator probe) are shown in Table <NUM> (SEQ ID NOS: <NUM>-<NUM>).

The absolute amount indicator may be a nucleic acid standard substance for quantitative analysis, which was developed by the National Institute of Advanced Industrial Science and Technology (AIST), or it may be newly designed. When the absolute amount indicator is to be designed, for example, the RANDBETWEEN function of software "EXCEL" (from MICROSOFT) may be used, which can randomly return X number (X is a given number) of integers of <NUM> to <NUM>. The resulting random integers may be linked in a row to give a X-digit number consisting only of <NUM> to <NUM> so as to obtain a large number of sequences having X bases of random ATGC by converting <NUM> to A, <NUM> to T, <NUM> to C and <NUM> to G.

From these sequences, only sequences that have the sum of G and T equal to the sum of A and T are picked out. The picked sequences are subjected to Blast search against database such as GenBank at NCBI so as to select less homologous sequences among organism-derived nucleic acids. Primer sequences are allowed to flank both ends of the sequence, thereby designing a sequence. Moreover, the designed sequence may suitably be linked for extension or partially removed for shortening.

In order to keep the reaction efficiency upon amplification reaction as constant as possible, the difference between the amplified base length of the bacterium targeted for detection and the amplified base length of the absolute amount indicator is preferably made small. For example, if the amplified product of the bacterium targeted for detection is about <NUM> bp, the amplified product of the absolute amount indicator is preferably made to be about <NUM> bp to <NUM> bp.

On the other hand, if the amplified chain length is to be confirmed by electrophoresis or the like after the amplification, the amplified product of the absolute amount indicator is designed to have a length that differs from the length of that of the bacterium targeted for detection so that the absolute amount indicator-derived amplified product can be detected at a location separated from the band for the bacterium targeted for detection, thereby confirming success/failure of the amplification reaction prior to hybridization.

If the absolute amount indicator is added prior to amplification reaction, it should be nucleic acids that can be amplified with a specific primer pair, in other words, it should possess a nucleotide sequence complementary to the primer pair, and it should possess a nucleotide sequence that is not possessed by any of the bacteria targeted for detection or the bacteria not targeted for detection in order to be detected through hybridization.

A specific primer means that the sequence targeted for amplification can be limited, where the primer pair is not necessary a single pair. If necessary, a multiplex technique that uses two or more primer pairs may also be applied. Examples of the primer pairs are shown in Table <NUM>. A primer pair for absolute amount indicator (SEQ ID NOS: <NUM> and <NUM>) and a primer pair for bacterial amplification (SEQ ID NOS: <NUM> and <NUM>) may be used.

The DNA chip of the present invention is defined in the claims and has a plurality of various probes described in Item <NUM> above arranged (mounted) on a substrate that serves as a support.

The substrate that serves as a support may be in any form of a flat plate (glass plate, resin plate, silicon plate, etc.), a stick, beads or the like. When a flat plate is used as the support, predetermined probes can be fixed thereon by types at predetermined intervals (spotting method, etc.; see <NPL>), etc.). Alternatively, predetermined probes can sequentially be synthesized thereon by types at specified positions (photolithography method, etc.; see <NPL>), etc.). Another preferable form of the support may be, for example, one that uses hollow fibers. When hollow fibers are used as a support, a preferable exemplary DNA chip (hereinafter referred to as a "fiber-type DNA chip") can be obtained by fixing predetermined probes in respective hollow fibers by types, bundling and fixing all of the hollow fibers, and then repeatedly cutting the fibers with respect to the longitudinal direction of the fibers. This DNA chip may also be referred to as a DNA chip that has nucleic acids fixed in through holes of a substrate, which is also referred to as a so-called "through-hole-type DNA chip" (see <CIT>, etc.).

The method for fixing probes onto a support is not limited, and any binding mode can be employed. Moreover, fixing is not limited to direct fixing onto the support. For example, a support may be pre-coated with a polymer such as polylysine so that probes may be fixed onto the treated support. Furthermore, when a tubular body such as hollow fibers is used as a support, the tubular body may be made to retain a gel-like substance so that probes can be fixed to the gel-like substance.

Hereinafter, a fiber-type DNA chip, one form of DNA chips, will be described in detail. The fiber-type DNA chip can be prepared, for example, through Steps (i)-(iv) below.

Examples of the material to be used as the hollow fiber preferably include, but are not limited to, a material described in <CIT>.

The hollow fibers are three-dimensionally arranged such that their lengths are equal in the longitudinal direction (Step (i)). The method employed for the arrangement may be, for example, a method in which a plurality of hollow fibers are arranged in parallel at predetermined intervals on a sheet-like material such as an adhesive sheet to form a sheet, which is thereafter wound up in spiral (see <CIT>), or a method in which two porous plates provided with a plurality of pores at predetermined intervals are layered such that the pores meet each other, where hollow fibers are passed through the pores and then the two porous plates are temporary fixed at a distance and a curable resin material is charged and cured around the hollow fibers between the two porous plates (see <CIT>).

The produced arranged body is embedded so that the arrangement is not disordered (Step (ii)). The method of embedment may preferably be a method in which a polyurethane resin, an epoxy resin or the like is poured into the gap between the fibers, a method in which the fibers are adhered to each other by heat welding, or a method likewise.

The hollow of each hollow fiber of the embedded arranged body is filled with a gel-precursor polymerizable solution (gel-forming solution) containing the probe to allow polymerization reaction in the hollow (Step (iii)). As a result, the hollow of each hollow fiber can retain the gel-like substance having the probe fixed thereto.

The gel-precursor polymerizable solution refers to a solution containing a reactive substance such as a gel-forming polymerizable monomer, where said monomer or the like can be polymerized/crosslinked so that the solution becomes a gel-like substance. Examples of such monomer include acrylamide, dimethylacrylamide, vinylpyrrolidone and methylene-bis-acrylamide. In this case, the solution may contain a polymerization initiator and the like.

After fixing the probes in the hollow fibers, the block body is cut into slices in a direction intersecting with (preferably perpendicular to) the longitudinal direction of the hollow fibers (Step (iv)). The resulting slice can be used as a DNA chip. The thickness of this DNA chip is preferably about <NUM> to <NUM>. The block body can be cut, for example, with a microtome, a laser or the like.

Preferable examples of the above-described fiber-type DNA chip include a DNA chip (Genopal™) manufactured by Mitsubishi Rayon Co.

In the fiber-type DNA chip, the probes are three-dimensionally arranged in the gel as described above so as to maintain a three-dimensional structure. Accordingly, the detection efficiency is enhanced as compared to a flat DNA chip that has probes bound onto a coated surface of a glass slide, and thus a highly sensitive and highly reproducible examination can be realized.

The number of types of probes arranged on the DNA chip is <NUM> types or less, preferably <NUM> types or less and more preferably <NUM> types or less per DNA chip. By limiting the number (types) of the arranged probes to some extent, the intraoral bacteria of interest can be detected with higher sensitivity. The types of the probes are distinguished by the nucleotide sequences. Therefore, usually, even probes that are derived from the same gene are specified as different types of probes even if just a single difference exists between the nucleotide sequences.

The method for measuring the number of intraoral bacteria is, for example, a method comprising the following steps.

Hereinafter, the steps of the measurement method will be described one by one in detail.

In this step, an intraoral sample (intraoral bacteria (bacterial group)) collected is used as a specimen, where the nucleic acids of the bacteria contained in the specimen are extracted. The specimen can be collected, for example, from a desired subject human or organism. The type of the intraoral sample collected is not particularly limited. For example, saliva, plaque (subgingival plaque, supragingival plaque), tongue fur, mousewash or the like may be used.

In the case where saliva is used as the intraoral sample, the method for collecting saliva is not particularly limited, and examples thereof include a method in which saliva is directly flowed into a container, and a method in which saliva is impregnated into a cotton swab or another paper-like product. A subject human may chew gum before the collection of saliva so that saliva can be easily collected.

When the collected sample is transported, it is preferred to employ a transport method in which the collected sample is put into an airtight container and frozen, or a transport method in which a cotton swab with a case is used, wherein the sample is collected using the cotton swab, which is put into the case.

The amount of saliva to be collected is not particularly limited and can be suitably selected depending on the method for detecting DNAs of bacteria present in the intraoral sample. For example, in the case where the gene DNA in a sample is detected using a DNA chip, the amount of saliva of a subject human is preferably <NUM> or more, and more preferably <NUM> or more.

Without limitation, the nucleic acids obtained from the specimen may be allowed to make direct contact with the DNA chip or the like, or a nucleotide sequence region desired may be amplified by PCR or the like so as to allow the amplified fragments thereof to make contact with the DNA chip or the like. Specifically, for example, the site desired to be amplified is preferably ribosome RNA (<NUM> rRNA) gene in chromosomal DNAs of the intraoral bacteria. PCR primers that can be used for amplification of this region may preferably be, for example, SEQ ID NOS: <NUM> and <NUM> shown in Table <NUM>. The amplification of the nucleic acids by the PCR method can be carried out according to a common method.

The extracted nucleic acids and the amplified fragments thereof in this step may suitably be labeled so as to be used in the detection process following hybridization. Specifically, a method in which the terminal of the PCR primer is labeled with a reporter dye, a method in which a reactive nucleotide analog is incorporated upon reverse transcription reaction, a method in which biotin-labeled nucleotides are incorporated, and the like may be contemplated. Furthermore, labeling can be carried out through reaction with a fluorescently labeled reagent after preparation. As the fluorescent reagent, for example, various kinds of reporter dyes (e.g., Cy5, Cy3, VIC, FAM, HEX, TET, fluorescein, FITC, TAMRA, Texas red, Yakima Yellow, etc.) can be used.

In this step, the nucleic acids or the amplified fragments thereof obtained in Step (I) are allowed to make contact with the probes or the DNA chip to be used in the present invention. Specifically, a hybridization solution containing said nucleic acids or the like is prepared so as to allow the nucleic acids or the like in said solution to bind (hybridize) to the probe mounted on the DNA chip. The hybridization solution can suitably be prepared using a buffer such as SDS or SSC according to a common method.

The hybridization reaction can be carried out by suitably setting reaction conditions (type, pH, temperature and the like of the buffer) such that the nucleic acids or the like in the hybridization solution can hybridize with the probe mounted on the DNA chip under stringent conditions.

After washing, the detected intensity of each spot is measured with a device that can detect the label of the nucleic acids or the like bound to the probe. For example, if the above-described nucleic acids or the like are fluorescently labeled, a fluorescence detector such as CRBIO (from Hitachi Software Engineering), arrayWoRx (from GE Healthcare), Affymetrix <NUM> Array Scanner (from Affymetrix), GenePix (from Axon Instruments), ScanArray (from PerkinElmer) or Genopal Reader (from Mitsubishi Rayon) can be used to measure the fluorescence intensity. In the case where the device is a fluorescence scanner, scanning can be performed by suitably adjusting, for example, the laser output and sensitivity of the detection section, whereas in the case where the device is a CCD camera type scanner, scanning can be performed by suitably adjusting the exposure time. A quantitative method based on the scan results can be performed with a quantification software. The quantification software is not particularly limited, and the average value, the median or the like of the fluorescence intensity of the spot can be used for quantification. Furthermore, considering the dimensional accuracy of the spot area of the DNA fragment and the like, adjustment is preferably performed upon quantification, for example, using the fluorescence intensity of the spot having no probe as background.

In this step, the number of bacteria of the bacterial species targeted for detection is calculated based on the fluorescence intensity obtained from the DNA chip (or probes) through the procedures of Step (I) and Step (II). For example, there is a method in which the SN ratio is expressed based on the fluorescence intensity of the probe for detecting the bacterium targeted for detection and the fluorescence intensity of the background. Alternatively, it may be preferable to conduct detections for each bacterium under a plurality of conditions by varying the bacterial chromosomal DNA concentration so as to acquire a conversion factor (standard curve) for each bacterium in advance for calculating the chromosomal DNA concentration based on the fluorescence intensity obtained under each concentration condition, by which chromosomal DNA concentrations can be calculated from fluorescence intensities obtained under the respective conditions.

In either case, a correction coefficient may be calculated for each DNA chip so that fluorescence intensities of an absolute amount indicator probe acquired by detection of a plurality of DNA chips will be constant. Accordingly, comparison can be made among the DNA chips by taking the correction coefficient into account for a fluorescence intensity of a bacterium targeted for detection from each DNA chip.

Hereinafter, the present invention will be described more specifically by way of examples, although the present invention should not be limited thereto.

In order to design DNA probes for detecting bacteria, attention was focused on V3 and V4 as the variable regions of <NUM> rRNA. Among the V3 and V4 regions, sequences specific to the respective bacteria were selected to design and prepare nucleotide sequences represented by SEQ ID NOS: <NUM>-<NUM>.

<NUM> pg of genomic DNA derived from the respective bacteria purchased from ATCC was used as a specimen to be measured to evaluate performance of the bacteria-specific probes designed/prepared in Example <NUM>.

PCR was conducted with the following reaction liquid composition and reaction conditions to amplify the sequences of the regions of <NUM> rRNA targeted for detection of the bacteria-derived genomic DNA. Amplification reaction was conducted with ProFlex (from Applied Biosystems) using Premix Ex Taq™ Hot Start Version (from Takara) as a PCR kit. Primer conditions shown below were used. The <NUM>'-terminal of the forward primer was labeled with Cy5 to label the terminal of the amplified product.

After heating at <NUM> for a minute, a total of <NUM> cycles of "dissociation at <NUM> (<NUM> sec), annealing at <NUM> (<NUM> sec) and synthesis at <NUM> (<NUM> sec)", and cooling at <NUM> were conducted to obtain an amplified product.

A through-hole-type DNA chip was produced in the same manner as the method described in Example <NUM> of <CIT> (method for detecting methylated DNA and/or unmethylated DNA).

In this regard, the mounted probes were probes having the sequence information represented by SEQ ID NOS: <NUM>-<NUM> and <NUM> in Table <NUM>.

The following solutions were mixed to prepare a hybridization solution.

Using an automatic hybridization/washing machine (model: AHF-<NUM>, Mitsubishi Rayon), <NUM>µL of the hybridization solution was allowed to make contact with the above-described DNA chip and allowed to hybridize therewith at <NUM> for <NUM> hours.

Following hybridization, the DNA chip was washed under the following conditions. Washing with <NUM>µL of a <NUM> Tris-HCl/<NUM> NaCl/<NUM>% Tween-<NUM> solution for <NUM> seconds was repeated for <NUM> times, followed by washing with <NUM>µL of <NUM> Tris-HCl/<NUM> NaCl for <NUM> seconds repeated for <NUM> times.

After washing was finished, each chip was transferred into a <NUM> Tris-HCl/<NUM> NaCl mixed solution at room temperature.

Following washing, the fluorescence intensity of each spot on the DNA chip was measured under the following conditions using Genopal Reader (model: GR-S1, Mitsubishi Rayon).

The background value (median of fluorescence intensities of spots having no probe mounted) was subtracted from the fluorescence intensity of the spot on which a probe for detecting a targeted bacterium was mounted to calculate the fluorescence intensity resulting from hybridization. As a result, as shown in Table <NUM>, the fluorescence intensity of the total amount indicator probe that plays a role as a positive control was recognized in every bacterium-derived genomic DNA, and validity of the reaction was confirmed. Moreover, in each bacterium probe of the present invention, the fluorescence intensity was obtained only with respect to a bacterium of interest. According to the above-described results, it was shown that each of the probes of the present invention has high specificity.

With the cooperation of five healthy adults, a saliva assessment test was conducted. The saliva of the five adults, i.e., Subjects A, B, C, D and E, were assessed.

Saliva was collected by placing γ-collection swab RI (Eiken Chemical) in the mouse for a minute. The γ-collection swab RI was immersed in water in a tube and left at room temperature for <NUM> minutes to elute the bacterial components. Thereafter, the γ-collection swab RI was removed and the tube was placed in a centrifuge. DNAs were extracted from the resulting pellets using DNeasy Blood & Tissue Kit (QIAGEN).

PCR and detection for DNA analysis were carried out under the same conditions as those in Example <NUM>. In this regard, the probes mounted on the DNA chip were probes having the sequence information represented by SEQ ID NOS: <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> in Table <NUM>.

The background value (median of fluorescence intensities of spots having no probe mounted) was subtracted from the fluorescence intensity of the spot on which a probe for detecting a targeted bacterium was mounted to calculate the fluorescence intensity resulting from hybridization.

In addition, like Example <NUM>, a plurality of bacterial DNAs whose concentrations were already known were assessed to make a standard curve and the fluorescence intensity was converted to the bacterial count in <NUM> of saliva. As a result, as shown in Table <NUM>, the amount of each of existing five types of bacteria of interest and the ratio of Streptococcus mutans to the bacterial count of Streptococci were successfully calculated.

According to the above-described results, many items, i.e., the amount of each of existing dental caries-related bacteria, Streptococcus mutans, Streptococcus sobrinus, Lactobacilli and Streptococci, and the ratio of Streptococcus mutans to the bacterial count of Streptococci in the oral cavity were successfully calculated simultaneously in a short time.

Specifically, according to the present invention, the respective bacterial counts of Lactobacilli, Streptococcus sobrinus and Streptococci can be simultaneously calculated in a short time by using a DNA chip that carries probes corresponding to DNA sequences specific to the respective bacteria.

Claim 1:
A DNA chip, which carries probes (a) and at least one probe from among a probe (b) and a probe (c):
(a) probes comprising a nucleic acid that hybridizes to <NUM> rRNA specific to each of one or more oral bacteria to be detected, wherein the probes are any of sequences (i) to (iii):
(i)
- each sequence selected from nucleotide sequences represented by SEQ ID NOs: <NUM>, <NUM>, <NUM>, and <NUM>,
- each sequence selected from nucleotide sequences represented by SEQ ID NOs: <NUM>-<NUM>, or
- each sequence selected from nucleotide sequences represented by SEQ ID NOs: <NUM>-<NUM>;
(ii) a complementary sequence of a sequence of (i); and
(iii) a sequence having at least <NUM>% sequence identity to a sequence of (i) or (ii);
(b) a total amount indicator probe; and
(c) an absolute amount indicator probe of one type or a plurality of types,
wherein the types of the probes are distinguished by the nucleotide sequences.