WO1997041253A1 - Procede de depistage de micro-organismes dans des melanges par amplification modulaire par reaction en chaine de la polymerase - Google Patents

Procede de depistage de micro-organismes dans des melanges par amplification modulaire par reaction en chaine de la polymerase Download PDF

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Publication number
WO1997041253A1
WO1997041253A1 PCT/EP1997/002179 EP9702179W WO9741253A1 WO 1997041253 A1 WO1997041253 A1 WO 1997041253A1 EP 9702179 W EP9702179 W EP 9702179W WO 9741253 A1 WO9741253 A1 WO 9741253A1
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WIPO (PCT)
Prior art keywords
hybridization
hybridization probe
amplification
detection
probes
Prior art date
Application number
PCT/EP1997/002179
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German (de)
English (en)
Inventor
Robert-Matthias Leiser
Jens Sperveslage
Original Assignee
Mira Diagnostica Gmbh
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Filing date
Publication date
Application filed by Mira Diagnostica Gmbh filed Critical Mira Diagnostica Gmbh
Priority to AU28884/97A priority Critical patent/AU2888497A/en
Publication of WO1997041253A1 publication Critical patent/WO1997041253A1/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
    • 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

Definitions

  • the invention relates to a method for the detection of one or more microorganisms in a sample which contains a large number of different microorganisms by means of molecular biological techniques, such as amplification reactions, and a compilation of components for carrying out the method.
  • the method according to the invention is referred to as a modular polymerase chain reaction (PCR).
  • Another disadvantage of known identification systems is that a large number of microorganisms remain undetected when examining complex samples because their culture requirements are unknown.
  • primers for diagnostic tasks are designed in order to directly detect organisms of interest to be able to.
  • sequence information obtained in this way primers for diagnostic tasks are designed in order to directly detect organisms of interest to be able to.
  • a molecular biological method for genome analysis is known as HLA typing. After amplification of the locus in question, the allelic state is characterized by hybridization with probes of selected specificity.
  • the technical problem on which the invention is based is to provide a method which permits a reliable determination of any microorganisms present in a sample, even if these are present in a mixture with various types of microorganisms.
  • the method should also be simple and inexpensive to carry out.
  • the HLA typing differs from the inventive method z. B. in that the former relates to eukaryotic gene sequences and the analysis of the gene sequence of an individual organism.
  • the method according to the invention is suitable for the detection of one or more microorganisms in a sample which contains a large number of different microorganisms. Molecular biological techniques such as amplification reactions are used.
  • At least one hybridization probe (A) which is able to display conserved nucleic acid sequences in the microorganism (s) of interest and at least one hybridization probe (B) which is able to display the less conserved nucleic acid sequences in the microorganism (s) of interest in is able to be added to the sample with the proviso that at least one hybridization probe of type (A) and type (B) must be present per microorganism of interest and the sample is in a state capable of hybridization.
  • the resulting hybridization pattern identifies the microorganism (s) of interest.
  • the method according to the invention is advantageous due to its modular structure.
  • a general PCR reaction which is identical for large groups of microorganisms, is linked to a subsequent detection by using taxon-specific sequences within the amplified DNA.
  • hybridization probes that correspond to microbial nucleic acid sequences of different degrees of conservation, the detection and identification of microorganisms that are in a mixed sample is made possible without a separation for the identification e.g. B. through single colony passages is necessary.
  • a combination of general germ determination based on highly conserved sequence sections and individually definable specification by less conserved sequence sections is carried out.
  • parts of the genetic information are preferably amplified in vitro using the hybridization probes as starter molecules (for example by PCR).
  • the hybridization probe (s) A are preferably used as starters for the amplification and the hybridization probe (s) B for detection.
  • the hybridization probe (s) B is also possible to use the hybridization probe (s) B as a starter for the amplification and the hybridization probe (s) A for the detection, the hybridization probe (s) A and B as a starter for the amplification and the hybridization probe (s) B for the detection or to use the hybridization probe (s) A and B as starter for the amplification and the hybridization probe (s) A for detection.
  • the advantage of this embodiment is that the desired combination of amplification and detection with the hybridization probes of narrower and broader specificity is achieved in this way.
  • Part of the method is advantageously carried out on a solid phase by binding part of the hybridization probes, the coupling of the corresponding hybridization probe (s) to the solid phase taking place after the amplification or that the coupling of the corresponding hybridization probe (s) to the solid phase before amplification and the amplification takes place at least partially on the solid phase.
  • the less conserved sequence (s), which corresponds to the hybridization probe (s) B are located between the conserved sequence regions, which correspond to the hybridization probe (s) (n) A corresponds (correspond) or the conserved sequence (s) corresponding to the hybridization probe (s) A is or are located between the less conserved sequence regions that the (the ) Hybridization probe (s) B corresponds (correspond). This is advantageous because an additional selection of the sequence-correct amplification is achieved.
  • the amplification according to the invention can be carried out simultaneously with a plurality of starter pairs on a plurality of target sequences simultaneously. This offers the advantage of simultaneously amplifying those microorganisms in a reaction for which there are no sufficiently matching hybridization probes as starters.
  • Ribosomal gene sequences have proven to be particularly suitable for use in the method according to the invention. Ribosomal gene sequences offer the advantage that they have highly conserved and less conserved sections in the preferred close neighborhood of interest here. Another advantage of using these gene sequences is that ribosomal genes exist in multiple copies per genome of microorganisms, resulting in a comparatively higher sensitivity of the assay according to the invention contributes.
  • Figure 1 illustrates the relationships described above in a schematic representation.
  • the AI and A2 probes hybridizing with highly conservative regions of a gene structure of rDNA hybridize with two different regions of a DNA structure of a prokaryote coding for 16S rRNA. These areas flank a region in which there are medium and low conserved areas, which in turn interact with the hybridization probes B1 and B2.
  • the amplification results in a detectable amount of analyzable substance which, in the event of a positive reaction, enables families and / or types of microorganisms to be distinguished.
  • the hybridization conditions can in each case be selected so stringently by temperature, ionic strength and other factors, in particular influencing the hydrogen bonding, that the specificity of the hybridization reaction (s) between the target sequence and the hybridization probes required for the statement of the method is ensured.
  • the person skilled in the art can determine how to select and set the stringency in detail using means known to him (cf. U. Wobus, "Isolation, Fractionation and Hybridization of Nucleic Acids", Akademie-Verlag, Berlin, 1981, 229 pages).
  • the hybridization probes are selected depending on the analytical task. Both general bacterial count determinations in combination with the detection of special species and the analysis and characterization of very complex samples are possible.
  • reaction vessels which contain various hybridization probes, preferably bound to a solid phase, which are combined in a modular manner in such a way that a large number of samples or different analytical questions on one or a few samples are examined in parallel and simultaneously.
  • a combination which contains components for carrying out the method according to the invention. These include, among other components, the hybridization probe (s) A and B.
  • a kit contains at least one hybridization probe A and / or B coupled to a carrier, e.g. B. a reaction vessel. These reaction vessels can be used as modules for similar analysis problems.
  • the combination according to the invention preferably also contains reagents for carrying out amplification reactions and / or components for the detection of amplificates.
  • reagents for carrying out amplification reactions and / or components for the detection of amplificates include reaction buffer, dNTP mix, water, enzymes, method descriptions, instructions for use, warnings and the like.
  • hybridization probes A with broadband specificity served for the amplification of all bacteria present in the test sample and corresponded to a highly conserved section of the ribosomal 16S rDNA according to Stackebrandt and Liesack (Handbook of New Bacterial Systematics, p 151-193, 1993):
  • the hybridization probes B with narrower specificity served as detection probes.
  • Escherichia coli AAC GUC GCA AGA CCA AAG Seq. ID # 3
  • Bacillus subtilis GGT TGT TTG AAC CGC ATG GTT Seq. ID # 4
  • the probes were biotinylated at the 5 'end for detection using an ELISA reader. As a result, a color change could be detected after adding a streptavidin-conjugated peroxidase (Soumet et al., BioTechniques 19: 792-796 (1995)).
  • the germs were first grown in buffered peptone water and then the DNA of all organisms was isolated from this mixture sample using a DNA isolation kit.
  • a primer (530r, see above) was covalently bound to the cavity of the CovaLink TM plate according to the instructions of the company Nunc.
  • EDC 1-ethyl-3- (3-dimethylaminopropylcarbodiimide
  • the wells were sucked dry and washed twice with 0.2 M NaOH, each with a 5-minute incubation.
  • the mixture was then washed twice with hybridization solution (6x standard saline citrate [SSC], 5x Denhardt's solution, 100 ⁇ g / ml sheared and denatured herring sperm DNA).
  • SSC standard saline citrate
  • 5x Denhardt's solution 100 ⁇ g / ml sheared and denatured herring sperm DNA.
  • the biotinylated hybridization probes were adjusted to a concentration of 0.1 nmol / L in hybridization solution and filled into the cavities in 100 ⁇ l aliquots.
  • the hybridization reaction ran at 37 ° C for 3 h.
  • three washing steps were carried out at 37 ° C. The first time with 2x SSC, 0.1% Tween 20 for 20 minutes.
  • the strepavidin-conjugated peroxidase was then added.
  • the peroxidase (Sigma Chemica, St. Louis. MO, USA) was diluted 1: 1000 in SPO solution (100 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.05% Tween 20). 100 ul of this dilution was added to each well. The plate was incubated at 37 ° C for 30 minutes. It was then washed three times with SPO solution.
  • TMB solution 1.5 mg / ml tetramethylbenzidine; Sigma Chemica
  • 25 mM citric acid 50 mM NaH 2 PO 4 , 0.03% H 2 0 2 , 10% dimethyl sulfoxide [DMSO] were used as the substrate. , pH 5.0) dissolved and added. After 45 minutes at 37 ° C, the reaction was stopped with 25 ul 2M H 2 S0 4 and measured at 450 nm on an ELISA reader.
  • the color changes to be observed in the individual cavities were the indicator for the presence in the test sample of the type of bacteria that corresponded to the added detection probe.
  • Example 2 Water samples and suspensions of industrial skimmed milk powders are checked for the presence of Staphylococcus s ⁇ p., Salmonella ssp. and Escherichia ssp. examined.
  • oligonucleotides with the following sequence were selected as hybridization probes A with broadband specificity:
  • amplicons with a length of approximately 460 bp are obtained in the prokaryotes analyzed.
  • the oligonucleotide 16SA2 was selected for coupling to the CovaLink TM plate and was used in the reaction mixture at a concentration of 0.06 ⁇ M, while the oligonucleotide 16SA1 was used in the reaction mixture at a concentration of 0.5 ⁇ M.
  • the following oligonucleotides served as hybridization probes B with narrower specificity for the detection:
  • Staphylococcus 5 - TGT GCA CAT CTT GAC GGT - 3 Seq. ID # 7 Salmonella: 5 - CTG GCA GGC TTG AGT CTT - 3 Seq. ID # 8 Escherichia: 5 - CTC ATT GAC GTT ACC CGC - 3 Seq. ID # 9
  • Example 2 The experimental conditions were identical to those of Example 1, except for the hybridization and washing temperatures in the detection. Here, hybridization and washing were carried out at 52 ° C. For this purpose, the bacteria were added to the samples both individually and in a mixture in different concentrations and concentration ratios (10 3 or 10 fi germs / ml). Table 2 below summarizes the approaches and results in a semi-quantitative form.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Wood Science & Technology (AREA)
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Abstract

Ce procédé permet de dépister et d'identifier les micro-organismes qui se trouvent dans un échantillon mélangé sans qu'il soit nécessaire de séparer les germes, par exemple en les faisant passer dans des colonies individuelles, avant de les identifier. Ce procédé met en oeuvre des techniques de biologie moléculaire. On détecte et on différencie les micro-organismes dans le mélange en les hybridant avec des sondes capables de reconnaître les sections à différents états de conservation de l'information génétique des micro-organismes en question. A cet effet, on associe un procédé général d'identification de germes à base de sections très bien conservées de la séquence et un procédé de spécification à caractères individuels définis à base de sections de la séquence moins bien conservées. Afin d'assurer un dépistage de sensibilité suffisante, on met de préférence en oeuvre une amplification de la section de l'acide nucléique, par exemple par réaction en chaîne de la polymérase.
PCT/EP1997/002179 1996-04-26 1997-04-26 Procede de depistage de micro-organismes dans des melanges par amplification modulaire par reaction en chaine de la polymerase WO1997041253A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU28884/97A AU2888497A (en) 1996-04-26 1997-04-26 Process for detecting micro-organisms in mixtures by modular pcr

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19616750A DE19616750A1 (de) 1996-04-26 1996-04-26 Verfahren zum Nachweis von Mikroorganismen in Gemischen
DE19616750.7 1996-04-26

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WO1997041253A1 true WO1997041253A1 (fr) 1997-11-06

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999022023A2 (fr) * 1997-10-29 1999-05-06 Mira Diagnostica Gmbh Procede de caracterisation de microorganismes
EP1322780A1 (fr) * 2000-07-27 2003-07-02 The Australian National University Sondes combinatoires et utilisations associees
EP1464710A3 (fr) * 2003-04-02 2004-12-22 Canon Kabushiki Kaisha Sonde et une série de sondes utilisé pour la détection des agents infectueux, un support, et une méthode de criblage genétique

Families Citing this family (5)

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Publication number Priority date Publication date Assignee Title
DE19945964A1 (de) * 1999-09-24 2001-04-05 Biotecon Diagnostics Gmbh Verfahren und Nukleinsäuren zum Nachweis von brauereirelevanten Mikroorganismen
DE19945916A1 (de) * 1999-09-24 2001-04-05 Biotecon Diagnostics Gmbh Nukleinsäuremoleküle zum Nachweis von Bakterien und phylogenetischen Einheiten von Bakterien
FR2811321A1 (fr) * 2000-07-04 2002-01-11 Bio Merieux Amplificateur d'une region ribonucleique cible d'un arn ribosomal 16s ou adn pour un tel arn d'une espece eubacterienne et detection de telles especes
DE102004063801A1 (de) * 2004-12-30 2006-07-13 Henkel Kgaa Verfahren zur Herstellung von Farbschutzwirkstoff-Granulaten
FR3106834A1 (fr) * 2020-01-30 2021-08-06 Ocean Diagnostics Nouveau procédé de PCR multiplexe et utilisation

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EP0528306A2 (fr) * 1991-08-15 1993-02-24 F. Hoffmann-La Roche Ag Amorce et sonde pour la détection de mycobactérium
US5494795A (en) * 1993-05-05 1996-02-27 The United States Of America As Represented By The Secretary Of The Navy Specific oligonucleotide primers for detection of pathogenic campylobacter bacteria by polymerase chain reaction

Patent Citations (3)

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WO1990011369A1 (fr) * 1989-03-22 1990-10-04 Cemu Bioteknik Ab Diagnostic en phase solide de conditions medicales
EP0528306A2 (fr) * 1991-08-15 1993-02-24 F. Hoffmann-La Roche Ag Amorce et sonde pour la détection de mycobactérium
US5494795A (en) * 1993-05-05 1996-02-27 The United States Of America As Represented By The Secretary Of The Navy Specific oligonucleotide primers for detection of pathogenic campylobacter bacteria by polymerase chain reaction

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Title
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BARRY T ET AL: "THE 16S/23S RIBOSOMAL SPACER REGION AS A TARGET FOR DNA PROBES TO IDENTIFY EUBACTERIA", PCR METHODS & APPLICATIONS, vol. 1, 1991, pages 51 - 56, XP000609831 *
EHRMANN M ET AL: "REVERSE DOT BLOT HYBRIDIZATION: A USEFUL METHOD FOR THE DIRECT IDENTIFICATION OF LACTIC ACID BACTERIA IN FERMENTED FOOD", FEMS MICROBIOLOGY LETTERS, 1994, pages 143 - 149, XP000612834 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999022023A2 (fr) * 1997-10-29 1999-05-06 Mira Diagnostica Gmbh Procede de caracterisation de microorganismes
WO1999022023A3 (fr) * 1997-10-29 1999-09-16 Mira Diagnostica Gmbh Procede de caracterisation de microorganismes
EP1322780A1 (fr) * 2000-07-27 2003-07-02 The Australian National University Sondes combinatoires et utilisations associees
EP1322780A4 (fr) * 2000-07-27 2005-08-03 Univ Australian Sondes combinatoires et utilisations associees
EP1464710A3 (fr) * 2003-04-02 2004-12-22 Canon Kabushiki Kaisha Sonde et une série de sondes utilisé pour la détection des agents infectueux, un support, et une méthode de criblage genétique
EP1717323A3 (fr) * 2003-04-02 2006-12-20 Canon Kabushiki Kaisha Sonde et une série de sondes utilisé pour la détection des agents infectueux, un support, et une méthode de criblage genétique
US8080381B2 (en) 2003-04-02 2011-12-20 Canon Kabushiki Kaisha Infectious etiologic agent detection probe and probe set, carrier, and genetic screening method
CN101768638B (zh) * 2003-04-02 2014-04-30 佳能株式会社 传染性病原体检测用探针和探针组以及载体和基因检查方法

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AU2888497A (en) 1997-11-19

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