WO2009005178A1 - Microréseau pour la détection et l'identification de microorganismes associés à des maladies périodontiques et procédé de diagnostic de maladies orales infectieuses à l'aide du microréseau - Google Patents

Microréseau pour la détection et l'identification de microorganismes associés à des maladies périodontiques et procédé de diagnostic de maladies orales infectieuses à l'aide du microréseau Download PDF

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WO2009005178A1
WO2009005178A1 PCT/KR2007/003246 KR2007003246W WO2009005178A1 WO 2009005178 A1 WO2009005178 A1 WO 2009005178A1 KR 2007003246 W KR2007003246 W KR 2007003246W WO 2009005178 A1 WO2009005178 A1 WO 2009005178A1
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seq
genus
spiecies
microarray
oligonucleotide
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Cheol Min Kim
Hyun Jung Jang
Young Mi Choi
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University Industry Cooperation Foundation of Pusan National University
GENEIN Co Ltd
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University Industry Cooperation Foundation of Pusan National University
GENEIN Co Ltd
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Priority to PCT/KR2007/003246 priority Critical patent/WO2009005178A1/fr
Priority to KR1020107000177A priority patent/KR20100061438A/ko
Publication of WO2009005178A1 publication Critical patent/WO2009005178A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to a method for detection and identification of periodontal disease-causing infectious bacteria and a method for diagnosis of oral disease using the same. More particularly, the present invention relates to a microarry comprising at least one of gram positive bacteria-specific and gram negative bacteria- specific oligonucleotides, periodontal disease-causing bacteria's genus-specific and species-specific oligonucleotides designed from the ITS target region which is hypervariable, and all kinds of bactera's universal oligonucleotides designed from 23S rDNA which is conserved, a method for detection and identification of periodontal disease-causing bacteria, and a kit for diagnosis of infectious oral disease using the microarray.
  • the oral cavity is like a gate that provide a human body with energy, so immediately reflects state of health.
  • oral bacteria causing oral disease make an invasion upon a blood vessel and arrive at other parts of body through blood, and cause heart disease, a stroke, glycosuria, premature birth, birth of underweight baby((Jin LJ, et al., Hong Kong Med J. 9:31-37(2003), Pennisi E, Science, 307:1899-
  • the periodontal disease among the oral disease is divided into gingivitis and periodontitis, and is inherent in about 7 ⁇ 16% of adults ((Tendera AB, Patnet application 10-2006-7017584(2006), Jin LJ, et al., Hong Kong Med J. 9:31-37(2003)). Like this, the periodontal disease is univeral disease but is called silent disease because the peaple feel so good before being in a bad state of health like shaking teeth.
  • the periodontal disease one of the most general infectious disease of human, destroys tissues supporting teeth including alveolar bone by bactera((Jin LJ, et al., Hong Kong Med J.
  • the periodontal disease has an effect on one more teeth, and develops when bactera in plaque cause an inflammation to gingiva.
  • the gingiva disease developing in early stage of the periodontal disease make gingiva swll up.
  • the periodontal disease differs from other infectious diseases because the periodontal disease develops when bacteria in the host is out of balance(Shay K, Clin Infect Dis. 34:1215-1223(2002)).
  • the pulp and periapical Diseases, and periodontal disease cause the oral disease and the periodontal disease play a importnat role in the change of body state((Kwak JS, et al., J Kor Acad Cons Dent, 30:335-343(2005)).
  • the influencial bacteria causing the paradenitis is reported as anaerobic bacteria((Bascones-Martinez A and Figuero-Ruiz E, Avances en Periodoncia, 3:111- 118(2005)). At least 500 species were identified in periodontal pockets. But Table 1 shows that only some species of bactera is related with the paradentitis((Slots J, J Periodontol. 75:1553-1565(2004)). Only 50% of bactera existing in the oral was successfully cultured and used to clarify if it is the cause of the oral disease.
  • Porphyromona s Prevotella Campylobacter Gram-negative gingivalis intermedia rectus enteric rods
  • the periodontal disease is divided into chronic periodontitis and aggressive periodontitis by the bactera. If the chronic periodontitis develops, spirochete increase and 90% anaerobic bacteria and 75% gram-negative bacteria exist in a dental plaque. Porphyromonas gingivalis, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Peptostreptococcus micros, Treponema denticola, Eubacterium nodatum, Prevotella intermedia, Tannerella forsythensis, Selenomonas noxia and so on belong to the causing bactria.
  • microarray tecnique makes many types of pathogenic bactera be analyzed at once as one microarray and many genes of many bactera be analyzed at once((Park H, et al., J Clin Microbiol. 43:1782-1788(2005), Warsen AE, Appl Environ Microbiol. 70:4216-4221(2004), Sergeev N, et al., Biosens Bioelectron. 20:684-98(2004)).
  • the micraarray can detect many types of species at once and be especially used in study associated with doouble infection and microbial ecology.
  • the microarray is the standard showing comprehensive characteristics((Smoot LM, et al., Adv Dent Res. 18:6-11(2005), Vianna ME, et al., Oral Microbiol Immunol. 20:253-258(2005), Kim JH, et al., J Kor Acad Cons Dent, 28:178-183(2003), Lee YJ, et al., J Kor Acad Cons Dent, 30: 409-422(2005)). Vianna ME et al.
  • microarray using target probe based on 16S rDNA base sequencethat to detect bacteria associated root canal, is hard to culture by traditional culturing method((Vianna ME, et al., Oral Microbiol Immunol. 20:253-258(2005)).
  • Smoot LM et al. used DNA microarray to show characteristics of oral microbial colony((Smoot LM, et al., Adv Dent Res. 18:6- 11(2005)).
  • the microarray can be used to detect and analyze not only many aerobic pathogenic bacteria but also oral microorganism and used as clinical medicine.
  • the choosing and designing of probe is important for detecting species of many pathogenic bactera at once. That is, selecting gene comprised of both genus- specific conserved region and species-specific hyvariable region as taget gene for detecting genus-specific and species-specific bacteria is important((Gilbert GL, Trends MoI Med. 8:280-287(2002)).
  • the polymorphism of target gene make molecular biological analysis speedy and perfect through speedy and perfect design((Park H, et al., J Clin Microbiol. 43:1782-1788(2005)).
  • the 16S rDNA of which base sequences are already opened through public nucleoteice database is selected as a target region to detect bacteria by using molecular biological analysis in many papers((Baker GC, et al., J Microbiol Methods,
  • the new detecting method using 23S rDNA base sequences can only detect some different bactera or some species belonging to one genus ((Anthony RM, et al., J Clin Microbiol, 38:781- 788(2000), Dunbar SA, et al., J Microbiol Methods, 53:245-252(2003), Mitterer G, et al., J Clin Microbiol, 42:1048-1057(2004)).
  • ITS internal transcribed spacer region
  • 16S rDNA base sequences
  • ITS exists between 16S rDNA and 23S rDNA and contains two characteristic regions: the conserved region differenciated in genus level and the hypervariable region differenciated in species level (Kuwahara T, et al., Microbiol Immunol. 45:191- 199(2001 ), Conrads G, et al., lnt J Syst Evol Microbiol. 52:493-499(2002), Park H, et al., J Clin Microbiol.
  • the hypervariable region is the proper target region for detecting bacteria species.
  • the present invention provides genus-specific and species-specific oligonucleotides for detecting periodontal disease-causing aerobic and anaerobic bacteria, targeting ITS having both hypervariable region and conserved region.
  • Another object of the present invention is to provide a microarray comprising the present oligonucleotides as probes for detection and diagnosing of periodontal disease-causing bacteria.
  • Another object of the present invention is to provide a microarray comprising the present oligonucleotides as probes for detection and diagnosing of oral disease-causing bacteria.
  • Another object of the present invention is to provide a method for identification and detection of oral disease including periodontal disease-causing bacteria by using the miroarray.
  • Another object of the present invention is to provide a kit for identification and detection of oral disease including periodontal disease-causing bacteria by using the microarray.
  • the present invention provides target DNAs for detection and identification of bacteria, comprising any one of one base sequence selected from SEQ ID Nos. 1 to 8 or its partial sequence.
  • target DNAs are designed from ITS regions of 6 kinds of bactera, which have importantance in a clinical study but their base sequnces have been unknown.
  • the target DNA can to be used for designing primer or probe detecting the bactera.
  • the present invention provides an oligonucleotide for gram positive-specific identification of microorganisms associated with periodontal diseases, comprising any one sequence selected from SEQ ID Nos. 10 to 11 or its complementary sequence.
  • the present invention provides an oligonucleotide for gram negative-specific identification of microorganisms associated with periodontal diseases, comprising a sequence of SEQ ID Nos. 12 or its complementary sequence.
  • the present invention provides an oligonucleotide for genus-specific identification of microorganisms associated with periodontal diseases, comprising any one of one base sequence selected from SEQ ID Nos. 13 to 32 or its complementary sequence.
  • the present invention provides an oligonucleotide for species-specific identification of microorganisms associated with periodontal diseases, comprising any one of one base sequence selected from SEQ ID Nos. 33 to 68 or its complementary sequence.
  • the oligonucleotides of the present invention are designed based on multiple sequence alignment of ITS (internal transcribed spacer) hypervariable sequences of bacteria.
  • the oligonucleotides have specificity to the gram positive and gram negative bacteria, and can be used as primers for PCR amplification or probes for hybridization in order to specifically detect genus and species of the bacteria.
  • the present invention provides a gram positive-specific and gram negative-specific probe set
  • PCT/KR2006/0002307 a genus-specific and species-specific probe set for detection of microorganisms associated with periodontal diseases, comprising more than one oligonucleotide selected from the above oligonucleotides.
  • the present invention provides a kit for diagnosing gram positive-specific and gram negative- specific microorganisms associated with periodontal diseases and genus-specific and species-specific microorganisms associated with periodontal diseases, comprising more than one oligonucleotide selected from the above oligonucleotides.
  • the oligonucleotides may be labeled with radioactive or non-radioactive labeling agent, the latter comprises conventional biotin, Dig(digoxigenin), FRET(fluorescence resonance energy transfer) and fluorescent dye
  • oligonucleotides can be used as primers or probes and the kit can comprise another primers for PCR amplification of the target DNA.
  • the present invention provides a microarray comprising more than one oligonucleotide selected from oligonucleotides for gram positive-specific and gram negative-specific detection and genus-specific and species-specific detection of microorganisms associated with periodontal diseases, as the probes attached on a support.
  • the universal oligonucleotide designed on the basis of 16S rDNA and 23S rDNA by Kim CM, et al and oligonucleotide designed for distinction of gram-positive and gram-negative bacteria by
  • the probes may be any materials having base sequence of the above oligonucleotides, preferably any one selected from a group consisting of DNA (Deoxyribose Nucleic Acid), RNA (Ribosse Nucleic Acid), and nucleic acid analogues selected from PNA (Peptide Nucleic Acid), LNA (Locked
  • nucleic acid analogues is stable to enzymes such as nuclease, has structurally specific interaction with base sequence, and has advantage of stability in heat. That is, a characteristic of the oligonucleotides is the base sequence. Because the characteristic of oligonucleotides is the base sequence hybridized with the target base, the oligonucleotides can include nucleic acid analogues having above base sequence.
  • the probes can be manufactured for sense or antisense of the oligonucleotides. Therefore, the oligonucleotides have base sequence of the SEQ ID Nos. or its complementary sequence.
  • the probes can further include QC probe for quality control of the microarray (Cheolmin Kim, et al., KR Pat. No. 10- 0590901(2006), Jang HJ, et al., J Clin Microbiol., 42:4181-4188(2004), Cheolmin Kim, et al., KR Pat. No. 10-0650162, (2006)).
  • the QC probe is an oligonucleotide which has a base sequence complementary to target sequences or any base sequence and which is labeled with a fluorescent material having an excitation/emission wavelength from that of a fluorescent material labeled on target.
  • the scanning of fluoresecent signal radioating before or after hybridization stage can illustrate whether probes are fixed to support or not and hybridized to target gene or not, and verify the formation and density of probes fixed to support and the hybridization between the probes and target gene without spectral interference.
  • the support may be made of any one selected from a group consisting of slide glass, plastic, membrane, semiconductive chip, silicon, gel, nano material, seramic, metal material and optical fiber or those mixture.
  • the microarray of the present invention can be manufactured using conventional method such as a pin microarray (Microarray printing technology, Don Rose, Ph.D., Cartesian Technologies, Inc., Anal Biochem, 320(2):281-91 (2003)), a ink jet (Nat Biotech, 18;438-441(2000), Bioconjug Chem,13(1 );97-103(2002)), photolithography (Cur Opinion Chem Biol, 2;404-410(1998), Nature genetics supplement, 21 :20-24(1999)) or a electric array method (Ann Biomed Eng. 20(4):423- 37(1992), Psychiatric Genetics, 12;181-192(2002)).
  • the microarray of the present invention may further comprise an extraction agent for isolating target DNA, a PCR kit containing primers for amplifying target gene, a hybridization reaction buffer, a washing solution for the un hybridized DNA, a cover slip, dyes, a washing solution for unbound dyes and a description sheet for the microarray, except for the microarray of the present invention.
  • the present invention provides a method for detection and identification of microorganisms associated with periodontal diseases, comprising the following steps: a) purifying nucleic acids from a sample; b) amplifying target DNA among the purified nucleic acid; c) hybridizing the amplified target DNA with probes of the microarray according to the present invention; and d) detecting signals generated from the formed hybrid.
  • the probe may further comprise the QC probe which can control the quility of all stages icuding hybridizing.
  • the step b) for amplifying target DNA can be performed using Hot-start PCR, Nested PCR, Multiplex PCR, RT- PCR (reverse transcripase PCR), DOP (degenerate oligonucleotide primer) PCR, Quantitive RT-PCR, In-Situ PCR, Micro PCR, modified PCR such as Lab-on a chip PCR and isothermal amplification method such as RCA (rolling circle amplification) as well as general PCR reaction.
  • the detection method of the present invention can be performed using probe amplification or signal amplification reaction such as tyramide signal amplification, nanoparticle probe, Raman-active dye and branched DNA((Karsten SL, et al., Nucleic Acids Res.
  • probe amplification or signal amplification reaction such as tyramide signal amplification, nanoparticle probe, Raman-active dye and branched DNA((Karsten SL, et al., Nucleic Acids Res.
  • the purifying step a) can be performed using conventional DNA or RNA purification method or kit.
  • the amplifying step b) can be performed using conventional PCR method.
  • the detection of the PCR product can be performed using conventional electrophoresis wih agarose gel.
  • the signal detecting step d) can be performed using conventional fluorescence scanner after binding with conventional dyes such as Cy5 or Cy3.
  • a method for simultaneously genotying and detecting more than one microorganisms associated with periodontal diseases selected from a group consisting of the following members: universal microorganisms(patent application No.04-68313, SEQ ID NO: 46)( SEQ ID NO: 9 in the present invention); gram positive microorganisms(PCT/KR2006/000237, SEQ ID NO: 1 to 2)( SEQ ID NO: 10 to 11 in the present invention) and gram negative microorganisms(PCT/KR2006/000237, SEQ ID NO: 3)( SEQ ID NO: 12 in the present invention); genus Actinobacillus (SEQ ID NO: 13 to 14) and spiecies Actinobacillus (SEQ ID NO: 33 to 34); genus Actinomyces (SEQ ID NO: 15 to 16) and spiecies Actinomyces (SEQ ID NO: 35 to 36); genus Campylobacter (S
  • Treponema SEQ ID NO: 30
  • spiecies Treponema SEQ ID NO: 59
  • genus Veillonella SEQ ID NO: 31
  • spiecies Veillonella SEQ ID NO: 60
  • genus Propionibacterium SEQ ID NO: 32
  • spiecies Propionibacterium SEQ ID NO: 61
  • spiecies Leptotrichia buccalis SEQ ID NO: 50
  • spiecies Tannerella forsythia SEQ ID NO: 57 to 58. Therefore, the present invention provides a method for detecting gram positive- specific and gram negative-specific detection of bateria associated with periodontal disease and a method for detecting one or more bacteria species simultaneously.
  • the present invention provides a method for detection of bateria associated with periodontal disease using SBE (Single base extension), Sequencing, RFLP (Restriction fragment length polymorphism), or REA (Restriction endonuclease analysis) based on difference of one base sequence by using the oligonucleotides which is designed for gram positive-specific and gram negative-specific detection of periodontal disease-causing bacteria and for genus-specific and species-specific detection of periodontal disease- causing bacteria.
  • SBE Single base extension
  • Sequencing Sequencing
  • RFLP Restriction fragment length polymorphism
  • REA Restriction endonuclease analysis
  • the method for detecting existence of periodontal disease-causing bacteria and identifying gram positive-specific, gram negative, genus-specific and species- specific bacteria using the microarray of the present invention comprises the following steps: a) if necessary, purifying nucleic acids from a cultured or clinical sample; b) if necessary, amplifying the target sequence of bacteria or its part using more than one proper primers; c) hybridizing the amplified target DNA with probes having a sense or antisense or complementary sequence of genus-specific and species-specific oligonucleotides and gram positive-specific and gram negative-specific oligonucleotides of periodontal disease-causing bacteria, disclosed in Tables 2 and 4; d) detecting signals generated from the formed hybrid; and e) predicting the existence of the periodontal disease-causing bacteria in the sample.
  • the probes in the c) step, have a diversity of probe composition and more than one probes.
  • the probes are optimized to simultaneously hybrid with its target region under the same hybrid and washing condition which can detect gram positive and gram negative bacteria and genus and species of the periodontal disease- causing bacteria at once.
  • the present invention provides a microarray comprising probes set for detecting gram positive-specific and gram negative-specific bacteria and genus and species of the periodontal disease-causing bacteria attached on the support, which can simultaneously detect gram positive and gram negative bacteria and identify genus and species of the bacteria as quickly and exactly as passible from only one experiment of a sample.
  • FIG. 1 shows the target region for detecting bacteria in biological sample and the location of primers for amplifying the target region and probes.
  • FIG. 1 shows the
  • ITS region comprising genus level conserved region and hypervariable region, the location of primers common to bacteria which can amplify the ITS region, the location of gram positive or gram negative-specific probes and genus-specific or species- specific probes of periodontal diseases-causing bacteria designed by present inventor.
  • Peptostreptococcus micro TAGTTTTATCACTGTTTTTTTGGTTCATATTTTTTT
  • Pantoea agglomerans GGGGAACCTGCGGTTGGATCACCTCCTTACCTGAAG
  • ITS ATACCTTCCCGCGCAGTGTCCACACAGATTGTCTGA : type 1 TAAAAAGTAACGAGCAGAAAAAACCACTACAGGCTT
  • ITS TTGTCTGATGAAAATGTAGCAGTAAAAAATCTCTGC : tyre 1 AGGCTTGTAGCTCAGGTGGTTAGAGCGC ⁇ CCCCTGA
  • ITS TTGTCTGATAGATGTAAAGAAGCAAGACGGCTGCGA : tyre 2 AGTCGCGACACCTCGTGTCCCCTTCGTCTAGCGGTT
  • New genus-specific oligonucleotides for detecting periodontal disease-causing bacteria developed in the present invention are as shown in Table 3. [Table 3] New probes for bacteria genus-specific detection of periodontal disease-
  • New species-specific oligonucleotides for detecting periodontal diseases- causing bacteria developed in the present invention are as shown in Table 4.
  • FIG. 1 shows location of tartet region, primers and probes used for amplifying aerobic and anaerobic periodontal disease-causing bacteria.
  • FIG. 2 shows results of PCR amplification of the target by primer designed for bacteria-specific base sequence.
  • FIG. 3 show a microarray comprising a probe set consisting of bacteria universal probes and genus-specific and eriodontal disease-specific probes for detecting bactera associated with periodontal disease, attached on a support.
  • FIG. 4 shows results of hybridization reaction of bacterial common probes and genus-specific and species-specific probes for Actinomyces viscosus and Peptostreptococcus micros among periodontal disease-causing bacteria.
  • FIG. 5 shows results of hybridization reaction of bacterial common probes and genus-specific and species-specific probes for Actinomyces viscosus and Peptostreptococcus micros among periodontal disease-causing bacteria.
  • FIG. 6 shows results of hybridization reaction of bacterial common probes and gram negative-probes and genus-specific and species-specific probes for Actinobacillus actinomycetemcomitans among periodontal disease-causing bacteria.
  • FIG. 7 shows a microarray comprising a probe set consisting of bacteria universal probes, gram-positive and gram-negative probes and genus-specific and species-specific probes attached on a support for more specific detection of the periodontal disease-causing bacteria, as one support.
  • FIG. 8 shows results of analyzing state of spot were analyzed with laser scanner after 1 :1 mixture of the QC probes and the negative control probes were fixed to the slide.
  • FIG. 9 shows results of hybridization reaction of bacterial common probes and gram negative-specific probes and genus-specific and speies-specific probes for Porphyromonas gingivalis of gram-negative bacteria related with the periodontal disease.
  • FIG. 10 shows results of hybridization reaction of bacterial common probes and gram negative-specific probes and genus-specific and speies-specific probes for Pantoea agglomerans among periodontal disease-causing bacteria.
  • Example 1 Incubation of Bacteria and isolation of Genomic DNA
  • ATCC American Type Culture Collection
  • KCTC Korean Collection for Tpe Cultures
  • the strains were selected in each culturing media under each culturing conditions according to manual provided by ATCC and KCTC. And the present inventors obtained many clinical specimens through network with General University Hospitals in pusan, Gyeongsangnam-do. From the cultured media, strain colonies were obtained with a white gold ear and input 1.ml tube, 10OuI of InstaGene matrix (Bio-Rad, USA) was added thereto and suspended, and reaction was performed at 56 0 C for 30 minutes in constant temperature bath.
  • the reactant was shook for 10 seconds, heated at 100 0 C for 8 min, shook again for 10 sec, centrifuged at 12,000 rpm for 3 min, recovered DNA.
  • the extracted DNA is storaged in a freezer at -20 0 C .
  • All probes used for detection of microorganisms associated with periodontal diseases is confirmed specificity of probes by multiple alignment and BLAST searching as selecting ITS target base sequence of microorganism associated with periodontal diseases published in Genbank.
  • the genus-specific probes of microorganism associated with periodontal diseases is only complementary in each genus, is selected from base sequence having very lower similarity to genus of other genus (length: 15-25 mer).
  • the species-specific probes is selected from base sequence having very lower similarity to polymorphism of other species.
  • Designed genus and specis-specific probes were standed for in Table 3 to Table 4. Above all probes can be used as not being limited in base sequence of Tabel 2 to Table 4 but being designed primers and probes consisted of base sequence comprising it.
  • Primer is designed as a state labeling biotin and Primer for DNA chip is designed as a state labeling biotin at 5'. PCR were carried out in follow condition.
  • Reaction composition is added to water to be 25ul of total volume after adding 10 0 C PCR buffer(100 mM KCI, 20 mM Tris HCI (pH 9.0), 15 mM MgCI 2 ) 5 /d, dNTP(deoxynucleoside triphosphates) mixture (dATP, dGTP, dTTP, and dCTP each 10 mM) 1 ⁇ &, forward and backward primers (each 10 pmole) each 1 ⁇ i, Taq polymerase (5 units//"*, QIAGEN, Inc., Valencia, USA) 0.2 ⁇ i, template DNA 4 ⁇ &.
  • 10 PCR buffer 100 mM KCI, 20 mM Tris HCI (pH 9.0), 15 mM MgCI 2 ) 5 /d
  • dNTP(deoxynucleoside triphosphates) mixture dATP, dGTP, dTTP, and dCTP each 10 mM
  • Reaction condition is denaturation at 94 0 C for 3 minutes, denaturation at 94 0 C for 1 minute.
  • Annealing reaction is carried out at 50 0 C for 1 minute, extension reaction is carried out at 72 0 C for 1 minute, and we repeated 30 times these process.
  • PCR products amplified through the procedure described in Exemple 3 were analyzed by performing A gel electrophoresis.
  • the base sequences of target bacteria not to be included in existing public nucleotide data base were acquired by analyzing.
  • FIG. 2 shows the PCR results by using a pair of primers amplifying the target sequence for the bacterial-specific detection.
  • FIG. 2 shows the PCR products in approximately 800-1500 bp that is amplified with the forward primer 1387F and 1525F((Patenet Application 04-68313(2004), by the present inventors) designed by using the 16SrDNA gene and the reverse primer 389R and 520R designed by using the 16SrDNA gene and analyzed by performing a gel electrophoresis.
  • lane M is 100bp DNA ladder as a standard maker of molecular weight
  • lane N negative control group.
  • FIG. 2(a) is the PCR result of Porphyromonas gingivalis; lane 2-6 for the mixture of 16S-1387F and 23S-520R, lane 4 for ATCC 33277, lane 5 for ATCC 49417, lane 2,3,6 for clinical specimen. And, lane 1 is mixture of 16S-1525F and 23S-389R ATCC 49417, as a smaller than lane 5.
  • ATCC 981 1 lane 2-5 for clinical specimen, lane 7-10 for Streptococcus sanguinis, lane 7 for ATCC 10556, lane 8-10 for clinical specimen, lane 1 1 for Treponema denticola ATCC 95404, lane 12 for Veillonella parvula ATCC 10790, lane 13 for clinical specimen, and lane 14 for Propionibactehum acnes ATCC 6919.
  • FIG. 2(d) lane 1 for Fusobacterium periodonticum ATCC 33693, lane 2-4 for clinical specimen F. periodonticum, lane 6 for Campylobacter concisus ATCC 33237, lane 7 ⁇ 8 for clinical specimen C. concisus, lane 9 for Enterococcus faecalis ATCC 19433, and lane 11 ⁇ 14 for Pantoea agglomerans.
  • the bacterial-specific PCR product are amplified by using each pair of specific primers, discriminating primaraily other microorganism such as human DNA and viral DNA. This enables a rapid and precise diagnosis and reduces a diagnostic cost.
  • the proes designed in Example 2 were synthesized to retain a dT spacer having amine-modification and C6-15 bases at the ⁇ '-terminus and 15-25 nucleotides.
  • the genus-specific and species-specific probes developed in the present invention are the base sequence 13 to 68 of table 3,4. All kinds of bactera-specific((Kim Cheol Min,
  • the target probes designed in Exemple 5 were diluted to 5-100 pmol, were added 1-100 pmol Qc probe designed in Exemple 6 and Micro-spotting solution or 3*
  • the probes were attached onto a slide glass substate by using a Microarryer(Cartesian Technologies, PLXSYS 7500 SQXL Microarryer, USA). Two spots per one kinds of probe were attached the supports, then, the resulting microarray was placed in a slide box at a room temperature for 24 hours or incubated with a dry oven at 50 0 C for about 5 hours to fix the probes.
  • a Microarryer Cartesian Technologies, PLXSYS 7500 SQXL Microarryer, USA. Two spots per one kinds of probe were attached the supports, then, the resulting microarray was placed in a slide box at a room temperature for 24 hours or incubated with a dry oven at 50 0 C for about 5 hours to fix the probes.
  • Example 9 Washing of unfixed probes In order to remove probes remained not to react onto the slide finished quility examination by Exemple 9, what is mentioned below was performed.
  • the microarray was washed out by using 0.2% SDS(sodium dodecyl sulfate) at a room temperate and then, washed by usig distilled water. After, the resulting microarray was put in sodium borohydride(NaBH4) for 5 minutes then, washed at 100 0 C . Again, the resulting microarray was washed out by using 0.2% SDS(sodium dodecyl sulfate) and distilled water then, surface of substrate was dried completely with the centrifuge.
  • Example 10 Hybridization
  • the biotin-labeled target products prepared in Example 3 were thermally treated to be denaturated in to single strands and cooled to 4.
  • reaction solution containing Cy5- streptavidin or Cy3- streptavidin(Amersham pharmacia biotech, USA) and 10ml or
  • Example 11 Washing of unspecific hybridized DNA
  • the cover slip was removed using a 2X SSC washing solution (300mm NaCI, 30mm Na-Citrate, pH 7.0), and the slide was washed with 2X SSC and then 0.2X SSC, followed by centrifugation to fully dry to the slide glass.
  • 2X SSC washing solution 300mm NaCI, 30mm Na-Citrate, pH 7.0
  • the hybridized result was scanned using a non-confocal laser scanner (GenePix 4000Am, Axon Instruments, USA) and analyzed by image analysis.
  • FIG. 3 show a microarray comprising a support, as a probe set, consisting of bacteria universal probes for detecting microorganism associated with periodontal diseases and genus-specific and species-specific probes.
  • the microarray can designe two and more microarrays as one support, so The microarray can analyze a number of samples for a short time simultaneously.
  • FIG. 4 shows results of hybridization reaction for Actinomyces viscosus and Peptostreptococcus micros using mircoarray, and shows scan image of bacteria common probes (Patent No. 04-68313 SEQ ID NO. 9) and Actinomyces genus- specific (SEQ ID No. 15, 16) and A. viscosus species-specific probes (SEQ ID No. 36) and Peptostreptococcus genus-specific (SEQ ID No. 24) and P. micros species- specific probes (SEQ ID No. 52, 53) when double infected by two bactera.
  • FIG. 5 shows results of hybridization reaction for Staphylococcus saprophyticus using mircoarray, and shows scan image of bacteria common probes (Patent No. 04- 68313 SEQ ID NO. 9) and Staphylococcus genus-specific (SEQ ID No. 26) and S. aureus species-specific (SEQ ID No. 56) probes.
  • FIG. 6 shows detection for the existence of periodontal diseases-causing bacteria, and shows microarray comprising a support, as a probe set, consisting of bacteria common probes, gram-positive and gram-negative probes and genus-specific and species-specific probes, and shows scan image of bacteria common probes (Patent No. 04-68313 SEQ ID NO. 9) and gram-negative-specific probes (PCT/KR2006/000237), (SEQ ID NO. 10) and Actinobacillus genus-specific probes (SEQ !D No. 13) and A. actinomycetemcomitans species-specific probes(SEQ ID No. 33).
  • FIG. 7 show a microarray comprising a support, as a probe set, consisting of bacteria universal probes for detecting bactera associated with periodontal disease (application No.04-68313), (SEQ ID No. 9) and gram-positive and gram-negative probes(PCT/KR2006/000237), (SEQ ID No. 10,1 1 ,12) and genus-specific and species- specific probes.
  • the microarrays can analyze a number of samples at once because two more than microarrays can be designed with one support.
  • FIG. 8 shows results of quility state of microarray using QC probes.
  • FIG. 9 shows results of hybridization reaction for Porphyromonas gingivalis using mircoarray checked the quility in Exemple 8, and shows scan image of bacteria common probes (Patent No. 04-68313 SEQ ID NO. 9) and gram negative-specific probes (PCT/KR2006/000237), (SEQ ID No. 10), Porphyromonas genus-specific probes (SEQ ID No. 25) and P. gingivalis species-specific probes (SEQ ID No. 55).
  • FIG. 10 shows results of hybridization reaction for Pantoea agglomerans using mircoarray, and shows scan image of bacteria common probes (Patent No. 04-68313 SEQ ID NO. 9) and gram negative-specific probes (PCT/KR2006/000237 SEQ ID NO. 10), Pantoea genus-specific probes (SEQ ID No. 23) and P. agglomerans species- specific probes (SEQ ID No. 51).
  • composition and layout of each probes can be changed because this is only tipical example of probes layout among new oligonucleotides designed in the present invention.
  • the present invention developed the miroarray and the diagnosis kit for detecting periodontal diseases-causing bacteria comprising any one selected from a group consisting of genus- and species-specific probes designed from ITS of base sequence hypervariable and conserved regions of the bacteria, and comfirmed its specificity.
  • the present invention can provide an antibiotics therapy for accurately removing infectious agent related to periodontal by detecting existence of periodontal -causing bacteria and identifying gram posive- and gram negative-bacteria and genus and species of the bacteria, at once.
  • the present invention can provide speedy and perfect microarry method, detect and care periodontal disease at the initial stage, and improve diverse diseases including Cardiovascular System disease caused by bacteria associated with periodontal disease.
  • microarray can be used for dianosis of oral microorganism because microarray can detect many microorganisms at once by using probe of high density.
  • Microarray makes many microoranisms detected at once. When dentist and clinical doctor detect pathogenic bacteria in oral, they can use microarray for improvement of diagnosis, prevention, and treatment.

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Abstract

La présente invention porte sur un procédé de détection et d'identification de bactéries infectieuses provoquant des maladies périodontiques et sur un procédé de diagnostic d'une maladie orale à l'aide de celui-ci. Plus particulièrement, l'invention porte sur un microréseau comprenant au moins l'un parmi les oligonucléotides spécifiques de bactéries à Gram positif et spécifiques de bactéries à Gram négatif, les oligonucléotides spécifiques d'un genre et spécifiques d'une espèce de bactéries provoquant une maladie périodontique mis au point à partir de la région cible ITS qui est hyper variable, et toutes les sortes d'oligonucléotides universelles de bactéries mises au point à partir d'ADNr 23S qui est conservé, sur un procédé de détection et d'identification de bactéries provoquant une maladie périodontique, et sur un coffret pour le diagnostic d'une maladie orale infectieuse à l'aide du microréseau. Conformément à la présente invention, celle-ci peut fournir une méthode à microréseau rapide et parfaite pour la détection d'une maladie périodontique à son stade initial. Par conséquent, diverses maladies systémiques comprenant la maladie du système cardiovasculaire provoquées par des bactéries associées à une maladie périodontique peuvent être améliorées.
PCT/KR2007/003246 2007-07-04 2007-07-04 Microréseau pour la détection et l'identification de microorganismes associés à des maladies périodontiques et procédé de diagnostic de maladies orales infectieuses à l'aide du microréseau Ceased WO2009005178A1 (fr)

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WO2010054445A1 (fr) * 2008-11-17 2010-05-20 Oral Health Australia Pty Ltd Détection d'infection par tannerella forsythia
US9714940B2 (en) 2010-10-22 2017-07-25 T2 Biosystems, Inc. NMR systems and methods for the rapid detection of analytes
US9046493B2 (en) 2010-10-22 2015-06-02 T2 Biosystems, Inc. NMR systems and methods for the rapid detection of analytes
US9702852B2 (en) 2010-10-22 2017-07-11 T2 Biosystems, Inc. NMR systems and methods for the rapid detection of analytes
US9360457B2 (en) 2010-10-22 2016-06-07 T2 Biosystems, Inc. NMR systems and methods for the rapid detection of analytes
US9488648B2 (en) 2010-10-22 2016-11-08 T2 Biosystems, Inc. NMR systems and methods for the rapid detection of analytes
US8883423B2 (en) 2010-10-22 2014-11-11 T2 Biosystems, Inc. NMR systems and methods for the rapid detection of analytes
US11098378B2 (en) 2012-04-20 2021-08-24 T2 Biosystems, Inc. Compositions and methods for detection of candida species
US9562271B2 (en) 2012-04-20 2017-02-07 T2 Biosystems, Inc. Compositions and methods for detection of Candida species
US10760109B2 (en) 2014-06-06 2020-09-01 The Regents Of The University Of Michigan Compositions and methods for characterizing and diagnosing periodontal disease
WO2015188178A1 (fr) * 2014-06-06 2015-12-10 The Regents Of The University Of Michigan Compositions et méthodes de caractérisation et de diagnostic d'une maladie parodontale
US11713478B2 (en) 2014-06-06 2023-08-01 The Regents Of The University Of Michigan Compositions and methods for characterizing and diagnosing periodontal disease
US11519016B2 (en) 2016-01-21 2022-12-06 T2 Biosystems, Inc. NMR methods and systems for the rapid detection of bacteria
CN109790531A (zh) * 2016-07-11 2019-05-21 三菱化学株式会社 口腔内检查方法
CN113528684A (zh) * 2021-08-04 2021-10-22 领航基因科技（杭州）有限公司 用于鉴别革兰氏阴性菌和革兰氏阳性菌的数字pcr检测试剂盒和应用
CN116334263A (zh) * 2023-04-21 2023-06-27 四川农业大学 一种用于检测鱼类鳃结节病病原菌的双重pcr引物、试剂盒及检测方法
CN118711657A (zh) * 2024-08-30 2024-09-27 杭州杰毅生物技术有限公司 一种ngs测序中针对标签跳跃污染的假阳性排除方法及系统

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