WO2001023609A2 - Sondes d'hybridation permettant de detecter des bacteries du genre thermobifida - Google Patents

Sondes d'hybridation permettant de detecter des bacteries du genre thermobifida Download PDF

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Publication number
WO2001023609A2
WO2001023609A2 PCT/EP2000/009461 EP0009461W WO0123609A2 WO 2001023609 A2 WO2001023609 A2 WO 2001023609A2 EP 0009461 W EP0009461 W EP 0009461W WO 0123609 A2 WO0123609 A2 WO 0123609A2
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probe
nucleic acid
thermobifida
bacteria
atcc
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PCT/EP2000/009461
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English (en)
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WO2001023609A3 (fr
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Olga Genilloud
Rafael P. Mellado
Victor Parro
Vicente Rodriguez
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Merck Sharp & Dohme De Espana, S.A.E.
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Publication of WO2001023609A2 publication Critical patent/WO2001023609A2/fr
Publication of WO2001023609A3 publication Critical patent/WO2001023609A3/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

Definitions

  • the present invention relates to nucleic acid probes which specifically detect strains of the genus Thermobifida (formerly classified as Thermomonospora). Said probes are derived from the region coding for the mature 16S ribosomal RNA (rRNA) of said strains. The invention also relates to assays using these probes.
  • nucleic acid probes made of genomic DNA, plasmids, riboprobes or synthetic oligonucleotides, are designed to target genomic DNA or certain RNA species present in biological samples.
  • the use of synthetic oligonucleotides as probes is largely preferred because oligonucleotides can be rapidly synthesized in large amounts using chemical methods, have a long shelf-life, and are easy to purify and label.
  • 16S and 23S rRNA genes are very often used for probe development given that sequences can readily be obtained using described methods and it is known that variable regions exist within these highly conserved genes which can be used for species-specific detection.
  • Species-specific probes have been described for a large number of microorganisms. Universal probes for the detection of bacteria are also known (Giovannoni, S.J. et al, 1988 J. Bacteriol 170:720-726, and Barry, T et al, 1991 supra).
  • This invention relates to nucleic acid probes which hybridize under hybridization conditions to nucleic acids encoding a portion of the 16S rRNA of bacteria belonging to the genus Thermobifida.
  • Another aspect of this invention is a method for detecting the presence of bacteria related to the genus Thermobifida, comprising:
  • kits for the detection of bacteria from the genus Thermobifida which comprises specific probes from 10 to 250 nucleotides in length which are complementary to or homologous with at least 90% of a nucleic acid sequence comprising Thermobifida nucleic acids corresponding to base pairs 430 to 445 of Thermobifida fusca DNA encoding the mature 16S rRNA molecule (Zhensui et al, 1998 Int. J. Syst. Bacteriol. 48:411-422).
  • the kit may additionally comprise reagents, compositions, instructions, disposable hardware and suitable packaging.
  • a method is provided herein which allows for the specific detection of bacteria from the genus Thermobifida.
  • the method makes use of nucleic acid probes that are complementary to a conserved region of the 16S rRNA genes present within the bacteria.
  • the probes herein provided permit rapid detection of bacterial strains of Thermobifida in an accurate and reproducible fashion.
  • the term "probe” will refer to synthetic or biologically produced nucleic acids, between 10 and 250 base pairs in length which contain specific nucleotide sequences that allow specific and preferential hybridization under predetermined conditions to target nucleic acid sequences, and optionally contain a moiety for detection or for enhancing assay performance.
  • Probes may optionally contain certain constituents that contribute to their proper or optimal functioning under certain assay conditions. For example, probes may be modified to improve their resistance to nuclease degradation (e.g., by end capping), to carry detection ligands (e.g., fluorescein, 32p ; biotin, etc.) or to facilitate their capture onto a solid support (e.g., poly-deoxyadenosine "tails").
  • detection ligands e.g., fluorescein, 32p ; biotin, etc.
  • Preferential hybridization or “hybridizing preferentially” means that hybridization with the intended target nucleic acid results in a hybridization reaction product which is more stable than any hybridization reaction product resulting from hybridization with a non-target nucleic acid under identical conditions. It is well within the skill of the ordinary artisan to compare stability of hybridization reaction products and evaluate which one is more stable, i.e., determine which one has bound "preferentially.”
  • homology and “homologous to” are meant to refer to the degree of similarity between two or more nucleic acid sequences and is not meant to imply any taxonomic relatedness between organisms.
  • the degree of similarity is expressed as a percentage, i.e., 90% homology between two sequences will mean that 90% of the bases of the first sequence are identically matched to the bases of the second sequence.
  • Specific means that a nucleotide sequence will hybridize to a predetermined target sequence and will not substantially hybridize to a non-target sequence.
  • Specific discriminate means that a probe will substantially hybridize to a predetermined target sequence and will not substantially hybridize to a non-target sequence.
  • Hybridization is a process by which, under predetermined reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion to form a double-stranded nucleic acid with specific and stable hydrogen bonds, following explicit rules pertaining to which nucleic acid bases may pair with one another.
  • Substantial hybridization means that the amount of hybridization observed will be such that one observing the results would consider the result positive in a clinical setting. Data which is considered “background noise” is not substantial hybridization.
  • Stringency hybridization conditions means approximately 35°C to 65°C in a salt solution of approximately 0.9 molar NaCl. Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and the temperature of hybridization. Generally as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a rule, the stringency of the conditions under which hybridization is to take place will dictate certain characteristics of the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art.
  • the probe In designing a probe for identification purposes, it is preferred that the probe be as specific as necessary (i.e., it should not cross-react with undesired nucleic acids) and highly sensitive (i.e., most, if not all, strains of the organism to be detected should react with the probe).
  • the preferred target sequences should have the following characteristics:
  • the sequence should be present in the genome of each strain of the microorganism concerned; and (b) the evolutionary diversity of the sequence should be such that, on the one hand, there is sufficient sequence-diversity to allow differentiation of the species concerned from other closely related species and, on the other hand, sufficient sequence-conservation to allow detection of the strain of concern with the probe used.
  • Probes were checked against all 16S rRNA available via Ribosomal Database Project World Wide Web server (Bonnie, L. et al, 1999 A new version of the RDP (Ribosomal Database Project), Nucleic Acids Res. 27:171-173).
  • RDP Ribosomal Database Project
  • two primers were designed. The first is designated BLT-N: 5' CCCAGACTCCTACGGGAGGCAGCAG 3' (SEQ ID NO:l) corresponding to base pairs 300-324 from Thermomonospora fusca DNA encoding mature 16S rRNA (Zhensui, Z et al, 1998 Int. J. Syst. Bacteriol. 48:411- 422).
  • the second is designated BIT-R: 5' CCGCCTACGAGCTCTTTACGCCCA 3' (SEQ ID NO: 2) corresponding to base pairs 526-549 from Thermomonospora fusca DNA encoding mature 16S rRNA (Zhensui et al, supra.). Both of these can be used as primers in polymerase chain reaction technology to amplify the DNA region in order to obtain a preferred probe of this invention. Designated large quantities of the probe can be generated using known PCR techniques such as those in U.S. patents 4,683,202 and 4,683,195.
  • a preferred probe CNB-THER.2 was derived from a variable region of the 16S rRNA molecule. It comprises the sequence 5' TTTCTGGGGGTTGACG 3' (SEQ ID NO:3), which is DNA corresponding to base pairs 430-445 from Thermomonospora fusca DNA encoding mature 16S rRNA.
  • probes similar to CNB-THER.2 may be made by increasing or decreasing the length of CNB-THER.2. For a longer probe, it is preferred that additional nucleotides (either 3' or 5') be those of the corresponding DNA of Thermomonospora fusca encoding mature 16S rRNA.
  • One embodiment of this invention is a nucleic acid, designated CNB-
  • THER.2 which provides specific binding to chromosomal DNA encoding the mature part of the 16S rRNA molecule of bacteria from the genus Thermobifida and does not substantially hybridize to the equivalent chromosomal region from bacteria belonging either to closely related actinomycetes taxa or to other bacteria.
  • the preferred probes of this invention generally contain from at least about 10 nucleotides to about 250 nucleotides (the maximum number of nucleotides of the mature region of the genes coding for the 16S rRNA).
  • the probe will contain from about 10 nucleotides to about 250 nucleotides resulting from the PCR amplification of a DNA fragment comprising a DNA sequence hybridizing with the 16 nucleotide-long CNB-THER.2 probe.
  • the invention also relates to probes for use in hybridization assays, which use an oligonucleotide sufficiently complementary to hybridize to a sequence of chromosomal DNA region encoding the mature 16S rRNA from bacteria of the genus Thermobifida, yet not complementary enough to hybridize to the equivalent region from far-related Gram positive bacteria (e.g., Bacillus subtilis).
  • a particularly preferred assay in accordance with this invention is a Southern blot.
  • One probe which can be used for a Southern blot assay is about 250 bp long, obtained by PCR amplification of the DNA fragment obtained by use of the two primers BLT-N and BIT-R.
  • the Southern blot, or dot blot assay can be conducted using well known procedures. Generally, it involves the steps of immobilizing a target nucleic acid or population of nucleic acids on a filter such as nitrocellulose, nylon or other derivatized membranes which are readily commercially available. The immobilized nucleic acids are then tested for hybridization under predetermined stringency conditions with the probe of interest. Under stringent conditions probes with nucleotide sequences with greater complementary to the target will exhibit a higher level of hybridization than probes whose sequences have less homology. Hybridization can be detected in a number of ways.
  • the probe can be isotopically labeled with the addition of a 32p_p osphorous moiety to the 5'-end of the oligonucleotide by the conventional polynucleotide kinase reaction. After hybridization has occurred, non-hybridized probe is removed by washing. The filters are exposed to x-ray film and the intensity of the hybridization signals is evaluated.
  • the probe CNB-THER.2 may be used as a primer in a Polymerase Chain Reaction in combination with the primer RVR.3 (SEQ ID NO:4, 5' AAGCTTTTAAGCAACATGCTCCGCCG 3 ') so as to specifically amplify a DNA fragment only when genomic DNA of the genus Thermobifida is used.
  • the temperature of annealing for specific detection is 50°C.
  • the probes of this invention may be chemically synthesized using commercially available methods and equipment.
  • the solid phase phosphoramidite methods can be used to produce short oligonucleotides between 15 and 30 nucleotides long.
  • short DNA oligonucleotides are synthesized chemically using any of the Applied Biosystems DNA Synthesizers with reagents supplied by the same company.
  • Chemically synthesized oligonucleotides can be obtained from Boehringer Mannheim and Perkin-Elmer. Strains which are detectable by the probes of this invention may be obtained from the ATCC collection and are the following:
  • Thermomonospora fusca (Thermobifida fusca comb, nov.)
  • ATCC 27730 Thermomonospora mesouviformis (Thermobifida mesouviformis comb. nov.) ATCC 27644
  • Other actinomycetes used in this invention also available from the ATCC collection are included in the following table:
  • stremptomycetes strains may be used in this invention: Streptomyces ambofaciens, S. antibioticus, S. cinnamonensis, S. coelicolor A3 (2), S. fradiae, S. lividans TK21, S. nataliensis, S. peucetius, S. violascens and Streptomyces sp.
  • Genomic DNA was prepared as follows: Approximately 0.5-1.0 g mycelia were resuspended in 2 ml lysis buffer (NaCl 0.1M, EDTA 50 mM, pH 8.0) containing glass beads (3 mm diameter) and the suspension was vortexed for 2 minutes before adding 2 ml of lysis buffer plus 10-15 mg lysozyme and 50 ⁇ g ml-1 Rnase Dnase-free. The suspension was incubated for 30-80 minutes at 37°C.
  • genomic DNA template Approximately 0.5-1.0 ⁇ g genomic DNA template was used with 280 ng of each primer in a final reaction volume of 100 ⁇ l of an incubation buffer containing 16.6 mM (NH_i)2SO4, 67 mM Tris-HCL (pH8.8), 0.1% Tween-20 and ImM MgCl2-
  • Amplifications were performed in automated thermocyclers by incubation at 95 °C (5 min) followed by 30 cycles of incubation at 95°C (1 min), 55°C (1 min) and 72°C (1 min) in the presence of one unit of Eco Taq polymerase (Ecogen).
  • the resulting about 250 bp long amplified DNA fragments were fractionated by electrophoresis in 1.5% agarose gels.
  • the fractionated DNA fragments were transferred to Hybond N+ membranes (Amersham, pic.) by capillary transfer for 16 h.
  • the DNA immobilized in the solid support was then washed with a hybridization buffer containing 5 x SSC, 5 x Denhardt's solution and 0.5% SDS and set to hybridize with 10 pmol of the radioactively labeled CNB-THER.2 probe in the same buffer for 16 h at 50°C.
  • the solid supports were then washed three times with 1 x SSC (0.15 M NaCl plus 0.015 M sodium citrate, pH 7.2) and 0.5% SDS at the hybridization temperature. The solid supports were then set to exposure in X-ray films at -80°C prior to being developed.
  • the CNB-THER.2 probe hybridized with the Thermobifida strains named above but it did not hybridize with any of the other actinomycetes mentioned. As expected, the probe also gave a negative result with low G+C content genomic DNA from the far-related bacterium Bacillus subtilis which served as a negative control.
  • EXAMPLE 2 Detection of Bacteria from the Genus Thermobifida by PCR
  • the probe CNB-THER.2 can be used as a forward primer for PCR in order to specifically amplify fragments from the target strains only.
  • As a reverse primer the conserved oligonucleotide RVR.3 (SEQ ID NO:4; 5' AAGCTTTTAAGCAACATGCTCCGCCG 3') can be used in all cases.
  • genomic DNA template of representative strains of each genera of bacteria listed above was used with 280 ng of each primer in a final reaction volume of 100 ⁇ l of an incubation buffer containing 16.6 mM (NH4)2SO4, 67 mM Tris-HCl (pH8.8), 0.1% Tween-20 and 1 mM MgCl2-
  • Amplifications were performed in automated thermocyclers by incubation at 95°C (5 min) followed by 30 cycles of incubation at 95°C (1 min), 50°C (1 min) and 72°C (1 min) in the presence of one unit of Eco Taq polymerase (Ecogen).
  • DNA fragments of the expected size were obtained only when the target organisms DNA were present in the reaction.

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Abstract

L'invention concerne une méthode permettant de détecter de manière spécifique des bactéries du genre Thermobifida. Cette méthode utilise des sondes d'acide nucléique complémentaires d'une région conservée des gènes d'ARN ribosomique 16S présents dans les bactéries. Ces probes permettent de détecter rapidement des souches bactériennes de Thermobifida de manière précise et reproductible.
PCT/EP2000/009461 1999-09-27 2000-09-25 Sondes d'hybridation permettant de detecter des bacteries du genre thermobifida WO2001023609A2 (fr)

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US15617299P 1999-09-27 1999-09-27
US60/156,172 1999-09-27

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999035285A2 (fr) * 1998-01-08 1999-07-15 Merck Sharp & Dohme De Espana, S.A.E. Sondes d'hybridation pouvant faire la differenciation entre le groupe de bacteries actinomadura madurae et les bacteries maduromycetes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999035285A2 (fr) * 1998-01-08 1999-07-15 Merck Sharp & Dohme De Espana, S.A.E. Sondes d'hybridation pouvant faire la differenciation entre le groupe de bacteries actinomadura madurae et les bacteries maduromycetes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BARRY T ET AL: "THE 16S/23S RIBOSOMAL SPACER REGION AS A TARGET FOR DNA PROBES TO IDENTIFY EUBACTERIA" PCR METHODS & APPLICATIONS,US,COLD SPRING HARBOR LABORATORY PRESS, vol. 1, 1991, pages 51-56, XP000609831 ISSN: 1054-9803 cited in the application *
GIOVANNONI S J ET AL.: "Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells" JOURNAL OF BACTERIOLOGY, vol. 170, no. 2, 1988, pages 720-726, XP000991116 cited in the application *
STACKEBRANDT ET AL: "Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes" APPLIED AND ENVIRONMENTAL MICROBIOLOGY,US,WASHINGTON,DC, vol. 57, no. 5, 1 May 1991 (1991-05-01), pages 1468-1477, XP002090717 ISSN: 0099-2240 *
ZHANG Z ET AL.: "Reclassification of Thermomonospora and Microtetraspora" INTERNATIONAL JOURNAL OF SYSTEMATIC BATERIOLOGY, vol. 48, 1998, pages 411-422, XP000991112 *

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