WO2003106676A1 - Sondes d'identification de micro-organismes et procede d'identification utilisant lesdites sondes - Google Patents

Sondes d'identification de micro-organismes et procede d'identification utilisant lesdites sondes Download PDF

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WO2003106676A1
WO2003106676A1 PCT/JP2003/007620 JP0307620W WO03106676A1 WO 2003106676 A1 WO2003106676 A1 WO 2003106676A1 JP 0307620 W JP0307620 W JP 0307620W WO 03106676 A1 WO03106676 A1 WO 03106676A1
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area
probe
region
identifying
detecting
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PCT/JP2003/007620
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English (en)
Japanese (ja)
Inventor
順也 橋田
上野　紳吾
勇 武藤
貴美子 成瀬
田村　美穂
耕一郎 松田
島津　光伸
寅喆 小林
石古　博昭
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日立ソフトウエアエンジニアリング株式会社
株式会社三菱化学ビーシーエル
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Priority to JP2004513489A priority Critical patent/JP4422019B2/ja
Publication of WO2003106676A1 publication Critical patent/WO2003106676A1/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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a specific probe for detecting and identifying microorganisms, particularly harmful bacteria in the fields of medicine and food, and a method for detecting and / or detecting the same using the same.
  • Microorganism Microorganism name Microorganism number
  • the first method is to use blood agar medium, MacConkey medium (for a stool, SS agar medium, etc.), various confirmation mediums, diagnostics, etc.
  • this method has a problem that the identification takes time.
  • PCR polymerase chain reaction
  • the region of 400-500 bases from the 5 'end of the 16S rRNA molecule is an effective region to distinguish between closely related species of the phylogenetically conserved genus, Although it has been reported that a probe having a partial nucleotide sequence of those regions is prepared to detect and identify a specific microorganism, the 16S rRNA nucleotide sequence has low conservation between microorganisms, The VI, V2 and V3 regions having high specificity for a particular species are not individually specified, and a probe is prepared from the nucleotide sequence of the region to efficiently and specifically identify the microorganisms shown in Table 1 of the present invention. No method has been reported for detection and identification. Disclosure of the invention
  • the present invention can quickly and reliably detect harmful microorganisms in the fields of medicine and food, based on the base sequences of the VI, V2 and V3 regions having high specificity for a specific species of 16S rRNA.
  • Another object of the present invention is to provide a probe for detecting and / or identifying a microorganism and a detection and / or identification method using the probe.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, focused on the base sequences of specific VI, V2 and V3 regions, which are particularly high in species, among 16S rRNA base sequences. However, they have found a probe that can specifically detect and / or identify harmful bacteria in the fields of medicine and food, and have completed the present invention.
  • the present invention provides the following 1 to 66.
  • Actinobacillus actinomycetemcomitans Actinobacillus actinomycetemcomitans, Acinetobacter calcoaceticus, Haemophilus influenza, Haemophilus ⁇ ⁇ uenzae, (Stenotrophomonas maltophilia) s Proteus 'Mirabilis (Proteus mirabilis), Streptococcus' Pneumoniae
  • Streptococcus oralis Streptococcus oralis, Staphylococcus aureus, Neisseria meningitidis, Capyronoctanae, Nanoha, Campylobacter fetus; Enterococcus gallinarumj, Enterococcus' caserifuranocus' (Enterococcus casselif lavus), Aeromonas' Nody drofira '(Aeromonas hydrophila), Sanoremonella' Noraffifi A (Salmonella paratyphi A), Sanoremonera phiphitostiphisti (Shiprepo typhisti strophy spell) equisimilis), Streptococcus canis, Streptococcus canis, Klebsiella oxytoca, Staphylococcus' Staphylococcus saprophyticus ⁇ Pasteurum munoletusida (Pasteu) rella multocida),
  • Mycobacterium avium Mycobacterium intracellulare (Mycobacterium intracellulare, Mycobacterium kansasi i), Mycobacterium gordonae (Mycobacterium gordonae)
  • the probe according to (1) which is selected from the nucleotide sequences represented by SEQ ID NOs: 1 to 152 or a complementary sequence thereof.
  • a probe for detecting and / or identifying Actinobacillus actinomycetemcomitans comprising the nucleotide sequence shown in SEQ ID NO: 1, 2, or 3, or a complementary sequence thereof.
  • a probe for detecting and / or identifying Hemophilus influenzae comprising the nucleotide sequence of SEQ ID NO: 7, 8 or 9, or a complementary sequence thereof.
  • a probe for detecting and / or identifying or identifying Stenotrophomonas maltophilia comprising the nucleotide sequence shown in SEQ ID NO: 10, 11, or 12, or a complementary sequence thereof.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 13, 14, or 15, or a complementary sequence thereof, for detecting and / or identifying Proteus mirabilis.
  • a probe for detecting and Z or identifying Streptococcus pneumoniae comprising the nucleotide sequence shown in SEQ ID NO: 16, 17, or 18, or a complementary sequence thereof.
  • a probe for detecting and / or identifying Pseudomonas aeruginosa comprising the nucleotide sequence shown in SEQ ID NO: 19, 20, or 21, or a complementary sequence thereof.
  • a probe for detecting and / or identifying Citrobacter frendi comprising the nucleotide sequence shown in SEQ ID NO: 22, 23 or 24, or a complementary sequence thereof.
  • a probe comprising the base sequence shown in SEQ ID NO: 25, 26 or 27, or a complementary sequence thereof, for detecting, identifying, or identifying Bayonella parvula.
  • a probe for detecting, identifying, or identifying Buffalo's stuarty comprising the nucleotide sequence shown in SEQ ID NO: 28, 29 or 30, or its complementary sequence.
  • a probe for detecting and / or identifying Neisseria gonorrhoeae comprising the nucleotide sequence shown in SEQ ID NO: 31, 32 or 33, or a complementary sequence thereof.
  • a probe for detecting and / or identifying Streptococcus agaratache comprising the nucleotide sequence shown in SEQ ID NO: 34, 35 or 36, or a complementary sequence thereof.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 37, 38 or 39, or a complementary sequence thereof, for detecting, detecting, or identifying Morganella 'morganii.
  • a probe for detecting, detecting, or identifying Pacteroides fragilis comprising the nucleotide sequence of SEQ ID NO: 40, 41 or 42, or a complementary sequence thereof.
  • a probe for detecting, Z or identifying Staphylococcus hominis comprising the nucleotide sequence shown in SEQ ID NO: 43, 44 or 45, or a complementary sequence thereof.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 49, 50 or 51, or a complementary sequence thereof, for detecting and / or identifying or identifying Staphylococcus hemolyticus.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 52, 23 or 53, or a complementary sequence thereof, for detecting and / or identifying Enterobacter.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 54, 55, or 56, or a complement thereof, for detecting and / or identifying Enterobacter aerogenes.
  • a probe for detecting and / or identifying Staphylococcus epidermidis comprising the nucleotide sequence shown in SEQ ID NO: 57, 58 or 59, or a complementary sequence thereof.
  • a probe for detecting and / or identifying Serratia marcescens comprising the nucleotide sequence shown in SEQ ID NO: 63, 23 or 64, or a complementary sequence thereof.
  • a probe for detecting, detecting, or identifying Streptococcus anginosus comprising the nucleotide sequence of SEQ ID NO: 65, 66, or 67, or a complementary sequence thereof.
  • a probe for detecting and / or identifying Escherichia coli comprising the base sequence shown in SEQ ID NO: 68, 69 or 70, or a complementary sequence thereof.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 54, 71 or 72, or a complementary sequence thereof, for detecting, detecting, or identifying Klebsiella pneumoniae.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 73, 74, or 75, or a complementary sequence thereof, for detecting, identifying, or identifying Enterococcus faecalis.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 76, 77, or 78, or a complementary sequence thereof, for detecting and / or identifying Enterococcus phenezim.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 82, 83 or 18, or a complementary sequence thereof, for detecting and identifying or identifying Streptococcus mitosis.
  • a probe for detecting and / or identifying or identifying Streptococcus intermedius comprising the nucleotide sequence of SEQ ID NO: 60, 84 or 67, or a complementary sequence thereof.
  • a probe for detecting and / or identifying Listeria monocytogenes comprising the nucleotide sequence shown in SEQ ID NO: 85, 86 or 87, or a complementary sequence thereof.
  • a probe for detecting, detecting, or identifying Clostridium perfringens comprising the nucleotide sequence of SEQ ID NO: 88, 89 or 90, or a complement thereof.
  • a probe for detecting, detecting, or identifying Corynepacteria aquatium comprising the base sequence shown in SEQ ID NO: 91, 92 or 93, or a complementary sequence thereof.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 94, 95, or 18, or a complementary sequence thereof, for detecting and Z or identifying Streptococcus' oralis.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 96, 97 or 98, or a complementary sequence thereof, for detecting and / or identifying Staphylococcus aureus.
  • a probe for detecting, detecting, or identifying Neisseria meningitidis comprising the nucleotide sequence of SEQ ID NO: 99, 100 or 101, or a complementary sequence thereof.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 102, 103 or 104, or a complementary sequence thereof, for detecting and / or identifying Campi pacter fetus.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 105, 106 or 107, or a complementary sequence thereof, for detecting and / or identifying Enterococcus' galinalum.
  • a probe for detecting and / or identifying Aeromonas' hydrofila comprising the nucleotide sequence shown in SEQ ID NO: 110, 111 or 112, or a complementary sequence thereof.
  • a probe for detecting and / or identifying Salmonella typhi comprising the nucleotide sequence of SEQ ID NO: 115, 114, or 53, or a complementary sequence thereof.
  • a probe for detecting and Z or identifying Streptococcus alleimiris comprising the nucleotide sequence shown in SEQ ID NO: 116, 117 or 118, or a complementary sequence thereof; 0
  • a probe comprising the nucleotide sequence of SEQ ID NO: 119, 120 or 121, or a complementary sequence thereof, for detecting and / or identifying S. caesus.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 52, 23 or 122, or a complementary sequence thereof, for detecting and / or identifying Klebsiella oxytoca.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 46, 123 or 124, or a sequence complementary thereto, for detecting and identifying or identifying Staphylococcus coccus saprofiticus.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 125, 126, or 127, or a complementary sequence thereof, for detecting and / or identifying Pasteurella multocida.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 128, 129 or 130, or a complementary sequence thereof, for detecting and / or identifying Eikenella corodense.
  • a probe for detecting and / or identifying Streptococcus pyogenes comprising the nucleotide sequence shown in SEQ ID NO: 131, 132 or 133, or a complementary sequence thereof.
  • a probe for detecting and Z or identifying Moraxella catarrhalis comprising the nucleotide sequence of SEQ ID NO: 134, 135 or 136, or a complementary sequence thereof.
  • a probe for detecting and / or identifying Legionella 'pneumophila comprising a nucleotide sequence represented by SEQ ID NO: 137, 138 or 139, or a complementary sequence thereof.
  • a probe for detecting, detecting, or identifying Mycobacterium tuberculosis cis comprising the nucleotide sequence of SEQ ID NO: 140, 141 or 142, or a complementary sequence thereof.
  • a probe for detecting and / or identifying Mycobacterium intracellulare comprising the nucleotide sequence shown in SEQ ID NO: 146, 147 or 145, or a complementary sequence thereof.
  • a probe for detecting and / or identifying Mycobacterium kansasii comprising the nucleotide sequence of SEQ ID NO: 148, 149 or 145, or a complementary sequence thereof.
  • a probe for detecting and / or identifying mycopacterium 'Gordone comprising the nucleotide sequence of SEQ ID NO: 150, 151 or 152, or a complement thereof.
  • a method for designing a probe comprising determining a mismatched site by comparing microorganisms, determining a region containing the mismatched site and having a base length of 20 to 100 bp.
  • One or more of the probes according to (1) or (2) is used, characterized by the fact that Actinobacillus actinomycetemcomitans, Acinetopactor '' Norecoaceticus, Hemofuinoles infnolenza, Stenotrophomonas ma Norrethophilia, Proteus 'Miravillis, Streptococcus pneumoniae, Syudomonas enoleginosa, Citrobatata Frendi, Bayonela Pal Pula, Providencia Stuati, Neisseria Gonoloye, Streptogastroe', Streptogastroe's-Mora Fragiris, Staphylococcus hominis, Staphylococcus penorenelli, Staphylococcus.
  • FIG. 1 is a diagram showing the VI, V2 and V3 regions in the base sequence of 16S rRNA of a microorganism. Description of Sequence Listing
  • SEQ ID NO: 153 is synthetic DNA.
  • SEQ ID NO: 154 is a synthetic DNA.
  • the probe for detecting and identifying microorganisms of the present invention and the method for detection and / or identification using the same will be described in more detail.
  • a first aspect of the present invention is a probe for detecting and / or identifying one or more microorganisms selected from the microorganisms shown in Table 1 above, wherein the probe comprises a microorganism to be detected and / or identified. It is a probe consisting of a base sequence of 20 to 100 bp in the VI, V2 and V3 regions of 16S rRNA or its complementary sequence.
  • a probe is an oligonucleotide DNA or RNA capable of detecting a specific fragment from DNA or RNA fragments by utilizing the property that complementary sequences of nucleic acids specifically bind to each other.
  • This is an oligonucleotide sequence that specifically binds to a target nucleic acid having a nucleotide sequence contained in 16S rRNA or 16S rDNA derived from a microorganism.
  • the probe capable of detecting and / or identifying each microorganism of the present invention is obtained by performing a multiple alignment between two or more microorganisms to be detected and identified in the VI, V2 and V3 regions of 16S rRNA of each microorganism, and In particular, the probe can be prepared by determining a region having high specificity for each microorganism (see FIG. 1).
  • the VI, V2, and V3 regions are the gene sequences of the 5 'region of 16S rRNA of each microorganism.
  • the VI region is the region of the 50th to 120th base from the 5 'end
  • the V2 region is the 150th to 260th base
  • the V3 region is a region of the 420th to 520th bases.
  • the base sequence of the probe is preferably 20 to 100 bp, particularly preferably 30 to 80 bp. Note that these probes are referred to as vl probe, v2 probe, and v3 probe according to each corresponding region.
  • probes may be partially modified, or may have deletions, substitutions, or additions in their nucleotide sequences, as long as the microorganisms shown in Table 1 can be specifically detected and identified.
  • VI, V2, and V3 regions of each microorganism were identified by homology search in the 16S rRNA base sequence of the microorganisms described in Table 1, and two or more microorganisms were identified in the VI, V2, and V3 regions.
  • the mismatch site is preferably designed near the center of the probe.
  • organisms with a minimum number of mismatches of 4 or more between the nucleotide sequence of the V1 to V3 region of the microorganism itself and the nucleotide sequence of vl to v3 probes derived from microorganisms other than the microorganism are: A microorganism that can be detected and identified by using a probe alone. Microorganisms with a minimum number of mismatches of 3 or less are microorganisms that can be detected and identified by comprehensively judging the results of detection using multiple probes.
  • Tables 2 and 3 show the IDs of the detection and identification probes for each microorganism and their sequence numbers.
  • Table 2 shows examples of probes that can identify microorganisms by themselves, and Table 3 shows examples of probes that can be identified in combination.
  • the probes capable of specifically detecting and identifying actinobacillus actinomycetemcommitans alone include the 01v2 probe having the base sequence shown in SEQ ID NO: 2 and the probe having SEQ ID NO: 3
  • the 01 v3 probe having the nucleotide sequence shown, or a probe having the complementary sequence thereof, and capable of specifically detecting and identifying Acinetobacter p. Calcoaceticus alone has the nucleotide sequence shown in SEQ ID NO: 4.
  • Table 3 shows microorganisms that are difficult to detect and identify using a single probe because the 16S rRNA gene sequence is similar to other microorganisms.
  • detection and identification of individual microorganisms can be performed using hybridization patterns of probes designed for the detection of other microorganisms as well as probes designed for the detection of the microorganism. Do. For example, as shown in Table 4, all of the probes 06 vl to v3 of SEQ ID NOs: 16 to 18 for detecting Streptococcus pneumoniae have the smallest mismatch between other microorganisms in the V1 to V3 region. Since the number is within 3 bases, it is difficult to detect and identify Streptococcus pneumoniae alone.
  • probes may be of natural origin or may be obtained by a conventional method such as chemical synthesis.
  • the chemical synthesis is performed by a phosphoramidite method using, for example, a DNA synthesizer of ABI (Applied Biosystem Inc.).
  • ABI Applied Biosystem Inc.
  • a second aspect of the present invention is a method for detecting and / or identifying one or more microorganisms selected from the microorganisms shown in Table 1, wherein the method comprises the steps of: A method characterized by using one or more probes each having a base sequence of 20 to 100 bp or a complementary sequence thereof in the V2 and V3 regions.
  • the detection and identification method of the present invention performs hybridization by bringing the probe into contact with a specimen containing a microorganism or a nucleic acid derived therefrom, and using the label as an index, the microorganism of Table 1 is used as an indicator. It is a method of detection and identification.
  • the specimen containing the microorganism or nucleic acid derived therefrom can be amplified using primers capable of amplifying the base sequence containing the V1 to V3 region of the 16S rDNA of the microorganism shown in Table 1.
  • a primer is a polynucleotide that acts as a starting point for the polynucleotide chain to elongate during a nucleic acid synthesis reaction.
  • the forward primer in the present invention is preserved between microorganisms upstream of the VI region.
  • the reverse primer is a region that is highly conserved between microorganisms downstream of the V3 region, approximately 450 to 620. It is preferable to design from the region existing at the ⁇ ⁇ th base.
  • the size is preferably 15 to 35 mer, particularly preferably 18 to 30 mer.
  • the foreprimer is located at about the 1st to 70th bases, the 27F primer shown in the following SEQ ID NO: 153, and the reverse primer is about the 450th to 620th bases. 525R shown in the following SEQ ID NO: 154 can be used.
  • These primers may be of natural origin or chemically synthesized by a conventional method.
  • the danigami synthesis can be synthesized, for example, by a phosphoramidite method using a DNA synthesizer manufactured by ABI (Applied Biosystem Inc.). It is also possible to use a known phosphate triester method, H-phosphonate method, phosphite method, or the like.
  • the nucleic acid includes RNA and DNA.
  • PCR can be achieved if the nucleic acid is present in a number of molecules to several tens of minutes or more.
  • Samples include, for example, stool, urine, blood, clinical test materials such as tissue homogenates, and food materials.
  • the extracted nucleic acid is made into type III, and a PCR method (Science 230, 1350 (1985)) is performed using the above primers.
  • the above primers (SEQ ID NOs: 153 and 154) are used to amplify the 27th to 525th bases from the 5 'end of the base sequence of the 16S rDNA of the microorganism and the sequences in the vicinity thereof. Is preferred.
  • Reaction conditions and reaction solutions for the PCR method can be arbitrarily set based on known information. For example, heat denaturation: 1 to 30 seconds at 90 to 95 ° C, annealing: 0 to 30 seconds at 37 to 65 ° C, extension reaction: 10 to 60 seconds at 50 to 75 ° C.
  • amplification is performed for 50 cycles.
  • the results of PCR amplification can be used to confirm the presence and length of amplified nucleotides by subjecting the solution after the reaction to agarol gel electrophoresis as needed.
  • the amplified nucleic acid of the microorganism can be used for hybridization using the above-described probe of the present invention.
  • hybridization means that, under specific conditions, two single-stranded nucleotide sequences having complementary sequences bind to each other to form a double strand.
  • the specific condition means a stringent condition in hybridization, and in particular, in the present invention, four or more mismatches between a base sequence derived from a microorganism and a base sequence of a probe of the present invention. In some cases, this means conditions that do not hybridize.
  • the stringency conditions vary depending on the hybridization conditions to be carried out, but those skilled in the art can use a solvent such as temperature, salt concentration, and activator concentration based on the hybridization protocol. Conditions such as composition can be appropriately set. As an example, hybridization with a 50-mer probe can be performed at 55 ° C., 0.5 ⁇ SSC, and 0.2% SDS. It should be noted that, depending on the purpose, it is possible to set the stringency to be higher so that the hybridization cannot be performed when the number of mismatches is 3 or more, 2 or more, or 1 or more. Also, lower stringency Thus, it is possible to set so that hybridization can be performed even when the number of mismatches is 5 or less, 6 or less.
  • hybridization is possible or not can be determined by extracting the amplified nucleic acid from the microorganism prepared as described above and labeling the amplified nucleic acid with a fluorescent substance such as fluorescein isothiocyanate / tetramethylrhodamine / isothiosinate / hapten, etc. After hybridization with the above-mentioned labeled nucleic acid, it can be confirmed by measuring a label such as a fluorescent dye. Labeling can be obtained by, for example, nick translation, a method using DNA polymerase, or nucleic acid amplification using a labeled primer whose 5 ′ end is labeled with a fluorescent substance or a hapten.
  • the detection and identification method of the present invention includes a method of detecting and identifying a specific microorganism shown in Table 2 by using a single probe. For example, the minimum mismatch between the nucleotide sequence of the probe consisting of the nucleotide sequence of 20 to:! OObp or its complementary sequence in the VI to V3 region of the microorganism to be identified and the nucleotide sequence derived from microorganisms other than the microorganism. Microorganisms can be detected and identified by using the above-mentioned probe having a number of 4 or more (see Tables 2 and 4).
  • the method of the present invention also includes a method of detecting and further identifying a specific microorganism shown in Table 3 by comprehensively judging the detection results obtained by the plurality of probes of the present invention.
  • a mismatch with the base sequence of a probe consisting of a 20 to 100 bp base sequence or its complementary sequence in the V1, V2 and V3 regions of the 16S rRNA of the microorganism to be identified is 3
  • Two or more microorganisms having a base sequence of not more than two or more are detected, and as a second step, the base sequence of 20 to:!
  • the method is to further identify one of the two or more microorganisms by using one or more probes having a complementary sequence thereof.
  • Example 3 an example is shown in Table 61 (see Example 3). Since there are three or less mismatches between the 06 vl-v3 probe of ID 06 microorganism (Streptococcus pneumoniae) and the base sequence corresponding to ID 29 microorganism (Streptococcus' miteisu),
  • ID 34 When the detection method is performed using a 34 vl to v3 probe derived from a microorganism (Streptococcus' Oralis), the ID06 microorganism hybridizes only with the 34 v3 probe and a signal is detected, and the ID29 microorganism is detected in 34 v2 and v3. The signal hybridizes with the probe and is detected.
  • the microorganisms detected using the 06vl-v3 probe can be further identified as ID06 microorganisms or ID29 microorganisms by performing detection using the 34vl-v3 probe derived from the ID34 microorganism (Streptococcus oralis). An organism can be identified.
  • the method of the present invention includes (a) a step of preparing a nucleic acid from a microorganism to be identified, (b) a step of hybridizing the nucleic acid with the probe of the present invention, and (c) the presence or absence of hybridization in the step (b). (D) identifying the detection signal pattern for each probe, (d) detecting the detection signal pattern obtained in step (c) and the detection signal pattern of the microorganisms specified in Table 1 specified in advance. A method comprising the step of identifying the type of microorganism to be identified by comparison is also included. In this case, by analyzing the detection signal pattern by computer processing or the like, the efficiency of detecting and identifying microorganisms can be further enhanced. The method using the detection signal pattern can be performed more quickly and effectively by performing the method on a DNA chip.
  • the detection and identification method on the DNA chip can be performed as follows.
  • the probe of the present invention is immobilized at each position (spot) on a support such as glass or silicon by a covalent bond or the like.
  • a solution containing the labeled nucleic acid obtained as described above is applied to the support, the base of the microorganism-derived nucleic acid in the sample having a base sequence complementary to the base sequence of the probe in each spot is obtained.
  • the sequences hybridize to form a double strand and remain on the support.
  • microorganisms shown in Table 1 were used as standard strains.
  • Microorganisms are distributed by the American Type Culture Collection (ATCC strain) and the Microorganisms Resource Division of the National Institute of Technology and Evaluation, National Institute of Technology and Evaluation (the Fermentation Research Institute (IF0)). (IF0), available from the Institute of Medical Science, The University of Tokyo (IID).
  • the VI, V2, and V3 regions of the microorganisms shown in Table 1 were determined by performing multiple alignment (Hitachi Soft DNASIS Pro) on the base sequence obtained by decoding 16S rRNA using a sequencer (Applied Biosysteni). Furthermore, using a so-called plast algorithm (Hitachi Soft DNASIS Pro), mismatch sites were identified in those regions. The nucleotide sequence of the probe was designed such that the mismatch site was near the center. Table 4 shows the number of mismatched bases for the v1 to v3 probes designed for each microorganism, assuming that they hybridize with the sequences of the V1 to V3 regions of microorganisms other than the microorganism from which each probe was derived.
  • the minimum value (minimum mismatch number). If the minimum number of mismatches in any of the v1 to v3 probes is 4 or more, the microorganism can be detected and identified by a single probe. In any of the vl to v3 probes, if the minimum number of mismatches is 3 or less, detection and identification cannot be performed by a single probe, but detection and identification can be performed by the method of the present invention as described later. In Table 4, none means that no sequence with significant homology was found. Table 4
  • vl v3 probe of the microorganisms shown in Table 1 was synthesized, purified by HPLC, and stored in a lyophilized state. These were adjusted to 20 ⁇ and mixed 1: 1 with a microarray spotting solution (Genetics). All samples were spotted into 2X4 blocks by a spotter (Hitachi Software SPBI0), and a total of 4X4 blocks including replicas were made. Positive controls were spotted on the four ends of each block.
  • the pin used in the spotter was a stainless steel pin 150 ⁇ . After spotting, the chip was placed in 0.2% SDS solution, water and boiling water, and the chip was washed with a slide washer (Piofield), and used in the following experiments.
  • the microorganisms shown in Table 1 were cultured on LB medium agar medium (5 g of yeast extract, 10 g of tryptone, 5 g of NaCl, 20 g of agar, 1 L of distilled water, pH 7.4) and then collected. Add 300 ⁇ l of 1% Tween20 (Sigma), Cell Suspension Solution (Gentra Systems) solution containing 60Unit Lytic Enzyme (Gentra Systems; 600Unit Achromopeptidase (Wako Pure Chemical)) (Bacterial Enzyme Solution), suspend and add at 37 ° C. Next, 30 ⁇ l of a 600 mU / ⁇ 1 Proteinase K solution was added, and the mixture was heated at 70 ° C. for 10 minutes.
  • Reaction solution 501 contains 11 type I DNA (microbial DM extraction solution), 5 ⁇ l 10X buffer (for Z-Taq), 0.75 units Z-Taq (Takara Shuzo), 6 nmole dNTPs Forward primer 27F (SEQ ID NO: 153) and reverse primer 525R (SEQ ID NO: 154), each containing 6 pmole.
  • the PCR conditions were as follows: after 2 minutes at 98 ° C, 1 cycle at 98 ° C and 90 seconds at 60 ° C, one cycle was performed and 35 cycles were performed to obtain a PCR amplification product of the target gene in the microorganism. After completion of the reaction, the substrate was removed by spin column method using Sephadex TM G50 Fine (Amersham pharmacia biotech), concentrated by lyophilization to dryness, and dissolved in sterile ultrapure water ( ⁇ ⁇ ) to obtain a target DNA solution.
  • the above target DNA was labeled with FluoroLink TM Cy5-dUTP (Amersham pharmacia biotech) using Nick Translation Kit (Roche Diagnostics).
  • FluoroLink TM Cy5-dUTP Amersham pharmacia biotech
  • Nick Translation Kit Roche Diagnostics
  • 20 ⁇ l of the labeling reaction solution 1 g of the above target DNA solution, 3.5 / l Enzyme mixture, 0.04 mM dATP, 0.04 mM dCTP, 0.04 mM dGTP, 21 lOx buffer, 0. 1 mM
  • FluoroLink TM Cy 5 ⁇ dUTP The reaction was performed at 15 ° C for 2 hours. After the reaction, purification was performed by a spin force ram method using Sephadex TM G50 Fine (Amersham pharmacia biotech). The purified reaction was lyophilized and dissolved in 10 / zl sterile ultrapure water.
  • the solution was dropped on the microorganism identification chip prepared in Example 1, and placed on a 24 ⁇ 25 mm high precover slide (Bhem Kiki Co., Ltd.), and reacted in a 55 ° C constant temperature bath for 2 hours. After the reaction, the bacteria identification chip was immersed in a 2x ssc solution to slide down the hyperica slide. In addition, 2 x SSC, 0.2 ° /. After transferring to an SDS solution and washing for 5 minutes, the cells were washed with 0.2 ⁇ SSC and 0.2% SDS for 5 minutes. Further washing was performed with 0.5x SSC for 1 minute.
  • Table 5 shows that the polynucleotide having the sequence containing the V1 to V3 region of the 16S rRNA of Actinobacillus actinomycetemcomitans of microorganism ID 01 is a vl probe of each microorganism prepared for the microorganism identification chip, v2 Probes and v3 probes were hybridized, and the top 10 probes with the highest signal brightness values were shown for each region.
  • the probe ID is a combination of the microorganism ID number shown in Table 1 and the vl to v3 regions.
  • the brightness of Probe 01 vl is remarkably high. It is possible to detect the presence of Nomycetem commitans.
  • the probe 03 vl other than the probe 01 vl has a similar luminance value, it cannot be identified by using the probe 01 vl alone. This is because the base sequence of the probe 01vl has three or less mismatches with the base sequence derived from another microorganism, so that it can be compared with other microorganisms having a base sequence highly similar to a similar probe 01vl. This is because they will hybridize (see Table 1). Therefore, when it is necessary to identify in detail which microorganism is the microorganism detected by the probe 01 vl, an additional identification step needs to be performed.
  • the probes with relatively high signal intensity in the v1 probe are 03 vl and 01 vl, and the signal intensity in the v2 probe is relatively high. Since the probe is 01 v2 and the v3 probe has a relatively strong signal intensity of 01 v3, the target microorganism was Actinobacillus' actinomycetes, where strong signal intensity was detected in all regions. It can be detected and identified as cetemcomitance. Table 5
  • Table 6 shows the 16S rRNA of A. pactor. Calcoaceticus (microorganism ID 02).
  • Table 7 shows the results of hybridizing a polynucleotide having a sequence containing the V1 to V3 region of the 16S rRNA of H. influenzae (microorganism ID 03) with each probe on the chip. From the results in Table 7, it is clear that Hemophilus'influenza can be detected and identified by using probes 03v2 and 03v3 alone. Also, even when comprehensively judged by the combination of the vl, v2, and v3 probes, 03 vl, 01 vl for the vl probe, 03 v2 for the v2 probe, and 03 v3 for the v3 probe have relatively strong signal strength. However, it can be detected and identified as Hemophilus' influenza, in which high intensity is detected in each region.
  • Table 8 shows the results obtained by hybridizing a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Stenotrophomonas maltophilia (microorganism ID 04) with each probe on the chip. From the results in Table 8, it is clear that Stenotrophomonas maltophila can be detected and identified by using probes 04 vl and 04 v3 alone. Also, even when comprehensively judged by the combination of vl, v2, and v3 probes, the signal intensity of 04 vl for the vl probe, 04 v2, 02 v2 for the v2 probe, and 04 v3 for the v3 probe has relatively strong signal strength. However, it can be detected and identified as a Stenot-mouth Fomonas maltophylla in which high intensity was detected in each region.
  • Table 9 shows the results obtained by hybridizing a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Proteus mirabilis (microorganism ID 05) with each probe on the chip.
  • V l even if the overall judgment in combination V 2, V 3 probe, vl probe in 05 vl, 01 vl, v2 in Puropu 05 v2, v3 in probe 05 v3, 45 v3 is relatively Since it has a strong signal intensity, it can be detected and identified as Proteus mirabilis in which high intensity was detected in each region.
  • Table 10 shows the results of hybridizing a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Streptococcus pneumoniae (microorganism ID 06) with each probe on the chip. From the results in Table 10, Streptococcus pneumoniae could not be identified by using probes 06vl to 06v3 alone. However, judging comprehensively by the combination of the vl, v2, and v3 probes, 06 vl, 29 vl for the vl probe, 06 v2, 29 v2 for the v2 probe, and 06_29_34 v3 for the v3 probe have relatively strong signal strength. It can be detected and identified as Streptococcus pneumoniae, in which high intensity was detected in each region.
  • Table 11 shows the results obtained by hybridizing a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Pseudomonas aeruginosa (microorganism ID 07) with each probe on the chip. From the results in Table 11, it is clear that Pseudomonas aeruginosa can be detected and identified by using probe 07 vl, 07 v2, and 07 v3 alone. Also, even when comprehensively judged by the combination of the vl, v2, and v3 probes, each signal has a relatively strong signal intensity of 07 vl for the vl probe, 07 v2 for the v2 probe, and 07 v3 for the v3 probe. It can be detected and identified as Pseudomonas aeruginosa with high intensity detected in the region.
  • Table 12 shows the results of hybridizing a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Citropactor 'Frendi (microorganism ID 08) with each probe on the chip. From the results in Table 12, it is clear that Citropactor'Frendi can be detected and identified by using Prop 08 v3 alone.
  • Table 13 shows the results obtained by hybridizing a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Bayonella pulpula (microorganism ID 09) with each probe on the chip. From the results in Table 13, it is clear that Bayonela 'pulpula can be detected and identified by using the probes 09vl, 09v2, and 09v3 alone. Also, even when comprehensively judged by the combination of the vl, v2, and v3 probes, the vl probe has a relatively strong signal intensity of 09 vl, the v2 probe has 09 v2, and the v3 probe has 09 v3. It can be detected and identified as bayonela / pulpura with detected intensity.
  • Table 14 shows the results obtained by hybridizing a polynucleotide having a sequence containing the VI to V3 regions of 16S rRNA of Buffalo Stuarty (microorganism ID 10) with each probe on the chip. From the results in Table 14, it is clear that Buffalo's quality can be detected and identified by using probes 10 vl, 10 v2, and 10 v3 alone. In addition, even when comprehensively judged by the combination of the vl, v2, and v3 probes, each region has relatively strong signal intensity of 10 vl, 13_vl for the vl probe, 10 v2 for the v2 probe, and 10 v3 for the v3 probe. Can be detected and identified as a Schau / Stuarty with a high intensity detected.
  • Table 15 shows the results of hybridizing a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Neisseria gonorrhoeae (microorganism ID 11) with each probe on the chip. From the results in Table 15, it is clear that Neisseria gonorrhoeae can be detected and identified by using probe 11 v3 alone.
  • vl, V 2, v3 even after comprehensive consideration a combination of probe, vl probe in 11 vl, 36 vl, v2 in probe 11 ⁇ 2, 36 v2, the v3 probe 11 v3 is relatively strong signal Because of the high intensity, it is possible to detect and identify Neisseria gonorrhoeae in which high intensity was detected in each region.
  • Table 16 shows the results obtained by hybridizing a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Streptococcus agaratache (microorganism ID 12) with each probe on the chip. From the results in Table 16, it is clear that Streptococcus agarakche can be detected and identified by using probes 12vl, 12v2, and 12v3 alone. Also, even when comprehensively judged by the combination of vl, v2, and v3 probes, 12 vl for the vl probe, 12 v2 for the v2 probe, and 12 v3 for the v3 probe have relatively high signal intensities, and are high in each region. It can be detected and identified as Streptococcus agalacti whose strength is detected.
  • Table 17 shows the results of hybridization of a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Morganella morga (microorganism ID 13) with each probe on the chip. From the results in Table 17, it is clear that Morganella morganii can be detected and identified by using probes 13v2 and 13v3 alone.
  • the vl probe has a relatively strong signal intensity of 13 vl, 10 vl, 13 v2 for the v2 probe, and 13 v3 for the v3 probe, It can be detected and identified as Morganella morganii, where high intensity was detected in the region.
  • Table 18 shows the results obtained by hybridizing a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Bacteroides fragilis (microorganism ID 14) with each probe on the chip. From the results in Table 18, it is clear that Pacteroides fragilis can be detected and identified by using Prop 14 vl, 14 v2, and 14 v3 alone. In addition, even when comprehensively judged by the combination of vl, v2, and v3 probes, 14 vl for the vl probe, 14 v2 for the v2 probe, and 14 v3 for the v3 probe have relatively strong signal intensities. It can be detected and identified as Pacteroides fragilis in which high intensity was detected in the region.
  • Table 19 shows the results of hybridizing a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Staphylococcus' hominis (microorganism ID 15) with each probe on the chip. From the results in Table 19, it was found that Staphylococcus 'Hominis' probe 15v :!
  • Table 20 shows the results obtained by hybridizing a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Staphylococcus' Perneri (microorganism ID 16) with each probe on the chip.
  • Table 21 shows the results of hybridization of a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Staphylococcus' hemolyticus (microorganism ID 17) with each probe on the chip. From the results in Table 21, Staphylococcus hemoriticus could not be identified by using probes 17 vl to 17 v3 alone, but when comprehensively judged by the combination of vl, v2, and v3 probes, 17 vl, 15 vl, 16_46 vl, 17 v2, 15 v2 for the v2 probe, and 17 v3 and 20 v3 for the v3 probe have relatively high signal intensities, so that high intensity was detected in each region. Was detected and identified.
  • Table 22 shows the results obtained by hybridizing a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Enterobacter 'croaka (microorganism ID 18) with each probe on the chip.
  • Table 23 shows the results obtained by hybridizing a polynucleotide having a sequence containing the VI-V3 region of 16S rRNA of Enterobacter aerogenes (microorganism ID 19) with each probe on the chip. From the results in Table 23, it is clear that Enterobacter aerogenes can be detected and identified by using probe 19 v3 alone. In addition, even when comprehensively judged by the combination of vl, v2, and v3 probes, relatively strong signal intensity is obtained at 19_25 vl for vl probe, 19 v2 for v2 probe, 08_18_22_45 v2, and 19 v3 for v3 probe. Enterobacter p. Aerogenes, whose high intensity was detected in each region, can be detected and identified.
  • Table 24 shows the results of hybridizing a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Staphylococcus' epidemidis (microorganism ID 20) with each probe on the chip. From the results in Table 24, Staphylococcus' epididermidis could not be identified by using probes 20 vl, 20 v2, and 20 v3 alone, but it was determined that the combination of the vl, v2, and v3 probes was comprehensive.
  • 20 vl, 15 vl for vl probe, 20 v2, 35 v2, 17 v2 for v2 probe, 20 v3, 46 v3, 17 v3, 16 v3, 15 v3 for v3 probe have relatively strong signal strength, It can be detected and identified as Staphylococcus 'Epidermidis' in which high intensity was detected in the region.
  • Table 25 shows the results obtained by hybridizing a polynucleotide having a sequence containing the 16S rRNA region of Streptococcus' consteratas (microorganism ID 21) with each probe on the chip. From the results in Table 25, it is clear that Streptococcus constellus can be detected and identified by using probe 21 v3 alone. Also, even when comprehensively judged by the combination of vl, v2, and v3 probes, 21_30 vl for vl probe, 30 v2, 21 v2 for v2 probe, and 21 v3 for v3 probe have relatively strong signal strength. It can be detected and identified as Streptococcus constellus where high intensity was detected in each region.
  • Table 26 shows the results of hybridization of a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Serratia marcescens (microorganism ID 22) with each probe on the chip. From the results in Table 26, it is clear that Serratia marcescens can be detected and identified by using probes 22vl and 22v3 alone. Also, even when comprehensively judged by the combination of the vl, v2, and v3 probes, the relative intensity of 22 vl for the vl probe, 08_18_22—45 v2 for the v2 probe, and 22 v3 for the v3 probe have relatively strong signal intensities. Serratia detected with high intensity in ''
  • Table 27 shows the results obtained by hybridizing a polynucleotide having a sequence containing the V1 to V3 region of the 16S rRNA of Streptococcus' anginosus (microorganism ID 23) with each probe on the chip. From the results in Table 27, it is clear that Streptococcus angiosus can be detected and identified by using probes 23vl and 23v2 alone. Also, even when comprehensively judged by the combination of the vl, v2, and v3 probes, each region has a relatively strong signal intensity of 23 vl for the vl probe, 23 v2 for the v2 probe, and 23-30 v3 for the v3 probe. Can be detected and identified as Streptococcus anguinosa with high intensity.
  • Table 28 shows the results obtained by hybridizing a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Escherichia coli (microorganism ID 24) with each probe on the chip. From the results in Table 28, it is clear that Escherichia coli can be detected and identified by using probe 24v3 alone.
  • 24v3 has a relatively strong signal intensity, it can be detected and identified as Escherichia coli in which high intensity was detected in each region.
  • Table 29 shows the results of hybridization of a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Klebsiella 'pneumoniae (microorganism ID 25) with each probe on the chip.
  • Table 30 shows the results of hybridizing a polynucleotide having a sequence containing the V1 to V3 region of 16S rRNA of Enterococcus ficalis (microorganism ID 26) with each probe on the chip. From the results in Table 30, it is clear that Enterococcus tenuis can be detected and identified by using probes 26 vl, 26 v2, and 26 v3 alone. Also, even when comprehensively judged by the combination of vl, v2, and v3 probes, 26 vl for the vl probe, 26 v2 for the v2 probe, and 26 v3 for the v3 probe have relatively strong signal intensities. It can be detected and identified as Enterococcus faecalis with high intensity.
  • Table 31 shows the results obtained by hybridizing a polynucleotide having a sequence containing the V1 to V3 region of the 16S rRNA of Enterococcus ′ fezium (microorganism ID 27) with each probe on the chip. From the results shown in Table 31, it is clear that Enterococcus faecium can be detected and identified by using probes 27vl and 27v2 alone. Also, even when comprehensively judged by the combination of vl, v2, and v3 probes, relatively high signal intensity is obtained at 27 vl for the vl probe, 27 v2 for the v2 probe, 27 v3, and 39 v3 for the v3 probe. However, it can be detected and identified as Enterococcus faecium in which high intensity was detected in each region.
  • Table 32 shows the results of hybridization of a polynucleotide having a sequence containing the VI to V3 region of 16S rRNA of Streptococcus sundaices (microorganism ID 28) with each probe on the chip. From the results in Table 32, it is clear that Streptococcus sanguis can be detected and identified by using probes 28 vl and 28 v2 alone.

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Abstract

L'invention vise à apporter des sondes spécifiques permettant de détecter et d'identifier un micro-organisme ainsi qu'un procédé d'identification et/ou de détection utilisant lesdites sondes. Les sondes de détection et d'identification d'un ou plusieurs micro-organismes nuisibles dans le domaine de la médecine, de l'alimentation, etc., notamment <i>l'Actinobacillus actinomycetemcomitans</i>, <i>Acinetobacter calcoeceticus</i> ou <i>Haemophilus influenzae</i>, comprennent entre 20 et 100 séquences de base bp dans les zones V1, V2 et/ou V3 de l'ARNr16 du micro-organisme à identifier ou de sa séquence complémentaire. Fait également l'objet de cette invention un procédé de détection et/ou d'identification d'un micro-organisme à l'aide d'une ou de plusieurs sondes.
PCT/JP2003/007620 2002-06-14 2003-06-16 Sondes d'identification de micro-organismes et procede d'identification utilisant lesdites sondes WO2003106676A1 (fr)

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