WO1999022023A2 - Procede de caracterisation de microorganismes - Google Patents

Procede de caracterisation de microorganismes Download PDF

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Publication number
WO1999022023A2
WO1999022023A2 PCT/EP1998/006863 EP9806863W WO9922023A2 WO 1999022023 A2 WO1999022023 A2 WO 1999022023A2 EP 9806863 W EP9806863 W EP 9806863W WO 9922023 A2 WO9922023 A2 WO 9922023A2
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WO
WIPO (PCT)
Prior art keywords
hybridization
microorganisms
substrate
seq
oligonucleotides
Prior art date
Application number
PCT/EP1998/006863
Other languages
German (de)
English (en)
Other versions
WO1999022023A3 (fr
Inventor
Matthias Leiser
Bernd Epping
Original Assignee
Mira Diagnostica Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mira Diagnostica Gmbh filed Critical Mira Diagnostica Gmbh
Priority to AU13370/99A priority Critical patent/AU1337099A/en
Publication of WO1999022023A2 publication Critical patent/WO1999022023A2/fr
Publication of WO1999022023A3 publication Critical patent/WO1999022023A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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 invention relates to a method for the detection of microorganisms of different taxa in a sample, which can contain a large number of different microorganisms.
  • microorganisms from complex samples (which contain several different germs in the mixture) is an important and difficult task, for example in hygiene examinations and other projects.
  • WO-A-97/41253 describes 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, at least one hybridization probe (A) being the preservative Able to display nucleic acid sequences in the microorganism (s) of interest and at least one hybridization probe (B) capable of displaying less conserved nucleic acid sequences in the microorganism (s) of interest is added to the sample with which Provided that at least one hybridization probe of type (A) and type (B) must be present for each microorganism of interest, the sample is in a state capable of hybridization and the resulting hybridization pattern is used to identify the microorganism (s) of interest.
  • a disadvantage of the described method is that many types of bacteria frequently show cross-reactions with the selected oligonucleotide sequences due to insufficient sequence variations in the area of the ribosomal genes, especially the 16S rDNA, and consequently cannot be differentiated from one another.
  • WO-A-96/00298 relates to a method for the simultaneous detection and identification and differentiation of Eu bacteria using a hybridization assay.
  • 16S-23S rR A spacer regions are amplified and the nucleic acids obtained are hybridized with probes of a specific type.
  • the disadvantage of this method is that the 16S-23S spacer region is not a functional section in many microorganisms and is therefore not subject to any or only a very low selection pressure.
  • differences in the lengths and sequences of the spacer regions can be detected even within one species in the various rDNA operons, which often reveal no relation to phylogenetic relationships (T.
  • EP-A-0 497 464 AI relates to a microbiological rapid assay by in situ hybridization in aqueous solution.
  • the microorganisms are initially included brought into contact with an aqueous composition, on the one hand fixing the microorganisms and on the other hand making the cell walls permeable to oligonucleotides.
  • labeled nucleic acid probes are added which are complementary to certain nucleotide sequences in the cellular nucleic acid, so that a hybridization reaction occurs, followed by detection of the hybridized nucleic acid.
  • This method relates to an in situ hybridization method.
  • the findings and methods cannot be transferred to in vitro analyzes as are customary for PCR and hybridization tests in reaction vessels with isolated target nucleic acid. In-situ methods have the disadvantage that sample preparation and / or test execution, particularly as far as the detection part is concerned, requires very complicated and expensive equipment.
  • US-A-5,614,361 relates to a method of characterizing an unknown organism in a sample by determining the position of some or all of the conserved DNA of the organism in relation to the position of restriction endonuclease cleavage sites in the DNA, a pattern characteristic of a particular microorganism sets.
  • a disadvantage of this method is that the identification of organisms on the basis of their DNA cleavage pattern after degradation by restriction endonucleases is very complex and requires the pure culture of the organisms to be analyzed.
  • the oligonucleotides according to the invention with the Seq. ID. No. 1 - 62 a practical bacterial identification according to the sequence listing.
  • variable regions of ribosomal gene sequences have been used frequently to establish phylogenetic relationships between different species and to design and use DNA probes or PCR start oligonucleotides for diagnostic or analytical purposes.
  • the method according to the invention also allows simultaneous detection and simultaneous detection of microorganisms of different taxa in a sample, which can contain a large number of different microorganisms.
  • tax means families, genera, species and subspecies of microorganisms.
  • the basis for the method according to the invention is the use of nucleic acid hybridization techniques.
  • Hybridization probes are used which interact with DNA or RNA, which are indicative of microorganisms and come from the organisms to be detected. If the concentration of nucleic acid to be detected is too low, amplification techniques can optionally be used to increase the concentration.
  • a hybridization result is obtained by hybridizing the probes with the DNA or RNA. It is essential according to the invention that at least one hybridization result is initially obtained for each microorganism to be detected. This can be done in particular by the method described in WO-A-97/41253 respectively. Reference is expressly made to the subject of WO-A-97/41253.
  • oligonucleotides are used as probes which are one of the sequences listed in the sequence listing with Seq. Id. Nos. 1 - 62 have identified sequences. According to the invention, both the entire ensemble of oligonucleotides with the numbers 1-62 can be used, or the user searches for the oligonucleotides associated with his detection problem with the assignment according to Table 1. It is essential to the invention that Table 1 indicates which of the oligonucleotide sequences mentioned is indicative of the taxon in question.
  • Acinetobacter anitra (DSM 30008), Acinetobacter baumannii (DSM 30007), Acinetobacter haemolyticus
  • Acinetobacter baumannii (DSM 30007), Acinetobacter baumannii (DSM 1139), Acinetobacter calcoaceticus (DSM 30006), Acinetobacter haemolyticus Aeromonas caviae, Aeromonas enteropelogenes Alcaligenes denitrificans, Alcaligenes faecalis Campylobacter species
  • Citrobacter freundii Edwardsieila tarda, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli / S.spp., Hafnia alvei, Klebsiella oxytoca, Plesiomonas shigelloides, Salmonella Typhimurium, Serratia marcescens, Vibrio vulnificus, Yersinia enterocolitica P Table 2 establishes the concordance between the oligonucleotides and their designation according to Table 1.
  • the starting oligonucleotide was 10-30f with the sequence
  • a further hybridization is preferably carried out by means of at least one hybridization probe which is different from the first hybridization probe, but also one of the Seq. ID No. 1-61. This then leads either to the occurrence or to the absence of cross-reactions. This Information can then be used to uniquely identify the microorganisms or a microorganism ensemble in a sample.
  • An advantage of the method according to the invention is therefore the possibility of clearly determining certain microorganisms alone or an ensemble of microorganisms simultaneously.
  • the hybridization results can then be evaluated, for example, by two-dimensional plotting of the hybridization results.
  • the result is a certain hybridization pattern, which is indicative of a certain microorganism ensemble.
  • Such patterns can e.g. stored in databases. In the case of serial examinations in particular, such patterns can be queried and used for quick and reliable identification.
  • the method according to the invention is therefore particularly suitable for automated investigations.
  • the cross-reaction is preferably detected spatially and / or temporally or by means of different markings on the probes.
  • the spatial arrangement of the hybridization results can preferably take place on a substrate.
  • Any carrier systems customary in molecular biology such as, for example, microtite laths, blotting papers, special membranes or DNA chips, are suitable as substrates.
  • a temporal dissolution of the cross reaction offers itself with flow-through methods.
  • Hybridization probes are added sequentially to the sample and their interaction with the nucleic acid contained in the sample is examined.
  • the hybridization results can be recorded in samples over time. For example, this is possible using dyes different spectral characteristics or fluorescent markers with short, different or staggered lifespans.
  • the hybridization patterns recorded as a function of time can also be stored in databases and then, when compared with a current sample, serve as an indication of the microbiological state of this sample.
  • the present invention therefore also relates to oligonucleotides with those in Seq. Id. Nos. 1-61 reproduced sequences.
  • the oligonucleotides according to the invention can be offered in particular in the form of kits together with aids for carrying out the method according to the invention.
  • the oligonucleotides are advantageously arranged on a substrate.
  • the arrangement can be made in particular in fixed assignments, so that in the case of positive hybridization, detection on a substrate appears in each case at a standardized, identical location, which facilitates automation of the evaluation.
  • sample material For pre-enrichment, 100 ml of the sample material is mixed with 300 ml of non-selective liquid medium (CASO broth) and incubated for 4 - 18 h in an incubator at 28 - 37 ° C (depending on the target organisms). Then 1.5 ml of this liquid enrichment are removed for the subsequent extraction of the DNA.
  • non-selective liquid medium CASO broth
  • This solution is at 8000 g for 2 min centrifuged, the resulting supernatant discarded and the sediment (pellet) in 100 ⁇ l buffer 1 (2 mg / ml lysozyme, 20 ⁇ g / ml lysostaphin, 100 mM Tris / HCl pH 7.2-7.4, 2 mM CaCl 2 , 4 % Sucrose solution, proteinase K solution (20 mg / ml H 2 O)) resuspended. Then 20 ⁇ l RNase A solution (20 mg / ml in sodium acetate buffer, pH 5.2) is added and the suspension is mixed on a shaker. The mixture is then incubated for 10 min at 60 ° C.
  • RNA cleaning columns QIAamp from QIAGEN.
  • the solution is mixed with 200 ⁇ l binding buffer (AL buffer QIAGEN) and 200 ⁇ l absolute alcohol and mixed well on the shaker.
  • the entire volume is then placed on a cleaning column (fixed in an empty tube) and centrifuged for 1 minute at 8000 g. The centrifugate is discarded, the cleaning column is placed in a new empty tube and charged with 500 ⁇ l washing buffer (AW buffer from QIAGEN).
  • Taq polymerase (5 U / ⁇ l) 0.2 ⁇ l
  • the target DNA is amplified in a thermal cycler (GeneAmp PCR System 9700 from Perkin Elmer) with a preferably heated lid.
  • the DNA is first denatured for 5 min at 94 ° C and then amplified for 30 cycles under the following conditions:
  • primer A can be used both for taxon-specific hybridization to a variable region of the target DNA and for broadband-specific hybridization to a sequence region homologous in all bacteria.
  • primer B hybridizes to a species-specific sequence region of the bacterial DNA and is therefore the decisive factor for determining the species / genus or group. For each The organism to be detected requires the starter oligonucleotides A and B required in each case.
  • the bacteria relevant for detection in milk are primarily the following genera / species:
  • DNA starter oligonucleotides listed below are used to identify the bacteria mentioned above:
  • E.coli 10-30f (Seq. ID No. 62) Es.co3r (ID No. 32)
  • Campylobacter species 10-30f Ca.jelr ID No. 20
  • Listeria species 10-30f Li.mo2r ID No. 41
  • Salmonella species 10-30f Sa.xx5r ID No. 53
  • DNA starter oligonucleotides listed below are used to identify the bacteria mentioned above:
  • Citrobacter freundii Edwardsieila tarda, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli / S.spp., Hafnia alvei, Klebsiella oxytoca, Plesiomonas shigelloides, Salmonella Typhimurium, Serratia marcescens, Vibrio vulnificus, Yersinia enterocolit
  • Example 4 For different samples occurring in practice, artificial mixtures were investigated in the experiment (Table 2)
  • the amplicons of the PCR reaction are applied to a 1% agarose gel and separated at 5-6 V / cm electrode spacing in an electric field by horizontal gel electrophoresis.
  • the agarose gel is then stained for 10 min in a 0.5 ⁇ g / ml ethidium bromide / TAE buffer solution and photo-documented at 254 nm under a UV transilluminator.
  • the developed starter oligonucleotides also detect the bacteria mentioned in a highly specific manner in mixed samples. If a sample contains one or more of the bacteria searched for, this is visible by a band of a defined size on the electrophoresis gel.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Procédé de caractérisation de microorganismes de différentes taxies, spécifiés au tableau 1, dans un échantillon pouvant contenir une pluralité de différents microorganismes de ces taxies, au moyen de techniques d'hybridation d'acides nucléiques, en utilisant des oligonucléotides de Séq. ID. No 1 à 62 comme sondes, tout en conservant un résultat d'hybridation, procédé dans lequel, pour chaque microorganismes à caractériser, au moins un résultat d'hybridation est maintenu.
PCT/EP1998/006863 1997-10-29 1998-10-29 Procede de caracterisation de microorganismes WO1999022023A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU13370/99A AU1337099A (en) 1997-10-29 1998-10-29 Method for identifying micro-organisms

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19747731.3 1997-10-29
DE19747731 1997-10-29

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WO1999022023A2 true WO1999022023A2 (fr) 1999-05-06
WO1999022023A3 WO1999022023A3 (fr) 1999-09-16

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001016363A2 (fr) * 1999-08-31 2001-03-08 GSF-Forschungszentrum für Umwelt und Gesundheit GmbH Detection precoce hautement specifique de pseudomonas aeruginosa par detection par sonde multiple
WO2001066797A2 (fr) * 2000-03-03 2001-09-13 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Systeme d'hybridation multiplex destine a l'identification de mycobacterium pathogene et procede d'utilisation
WO2001023605A3 (fr) * 1999-09-24 2002-02-21 Biotecon Diagnostics Gmbh Procede et acides nucleiques servant a determiner la presence de micro-organismes presentant un interet dans le domaine de la brasserie
WO2002095066A2 (fr) * 2001-05-18 2002-11-28 Biotecon Diagnostics Gmbh Detection de microorganismes de l'espece yersinia pestis/yersinia pseudotuberculosis et/ou differenciation entre yersinia pestis et yersinia pseudotuberculosis
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
US7070935B2 (en) 2000-02-18 2006-07-04 Science Applications International Corporation Method for detecting a biological entity in a sample
US7455966B1 (en) 2000-05-01 2008-11-25 Science Applications International Corporation System and method for detecting a biological entity in a water sample
EP2446062A2 (fr) * 2009-06-26 2012-05-02 The Regents of the University of California Procédés et systèmes d'analyse phylogénétique
US8771940B2 (en) 2006-11-30 2014-07-08 The Regents Of The University Of California Array for detecting microbes
US20140200149A1 (en) * 2012-03-06 2014-07-17 The Regents Of The University Of California Methods and compositions for identification of source of microbial contamination in a sample

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001016363A2 (fr) * 1999-08-31 2001-03-08 GSF-Forschungszentrum für Umwelt und Gesundheit GmbH Detection precoce hautement specifique de pseudomonas aeruginosa par detection par sonde multiple
WO2001016363A3 (fr) * 1999-08-31 2001-11-01 Gsf Forschungszentrum Umwelt Detection precoce hautement specifique de pseudomonas aeruginosa par detection par sonde multiple
US7183085B1 (en) 1999-09-24 2007-02-27 Biotecon Diagnostics Gmbh Method and nucleic acids for determining the presence of micro-organisms specific to the brewing process
WO2001023605A3 (fr) * 1999-09-24 2002-02-21 Biotecon Diagnostics Gmbh Procede et acides nucleiques servant a determiner la presence de micro-organismes presentant un interet dans le domaine de la brasserie
US7070935B2 (en) 2000-02-18 2006-07-04 Science Applications International Corporation Method for detecting a biological entity in a sample
WO2001066797A3 (fr) * 2000-03-03 2003-02-27 Us Gov Health & Human Serv Systeme d'hybridation multiplex destine a l'identification de mycobacterium pathogene et procede d'utilisation
WO2001066797A2 (fr) * 2000-03-03 2001-09-13 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Systeme d'hybridation multiplex destine a l'identification de mycobacterium pathogene et procede d'utilisation
US7271781B2 (en) 2000-03-03 2007-09-18 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Multiplex hybridization system for identification of pathogenic mycobacterium and method of use
US7455966B1 (en) 2000-05-01 2008-11-25 Science Applications International Corporation System and method for detecting a biological entity in a water sample
EP1322780A4 (fr) * 2000-07-27 2005-08-03 Univ Australian Sondes combinatoires et utilisations associees
EP1322780A1 (fr) * 2000-07-27 2003-07-02 The Australian National University Sondes combinatoires et utilisations associees
WO2002095066A2 (fr) * 2001-05-18 2002-11-28 Biotecon Diagnostics Gmbh Detection de microorganismes de l'espece yersinia pestis/yersinia pseudotuberculosis et/ou differenciation entre yersinia pestis et yersinia pseudotuberculosis
WO2002095066A3 (fr) * 2001-05-18 2003-12-04 Biotecon Diagnostics Gmbh Detection de microorganismes de l'espece yersinia pestis/yersinia pseudotuberculosis et/ou differenciation entre yersinia pestis et yersinia pseudotuberculosis
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
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
US8080381B2 (en) 2003-04-02 2011-12-20 Canon Kabushiki Kaisha Infectious etiologic agent detection probe and probe set, carrier, and genetic screening method
US8771940B2 (en) 2006-11-30 2014-07-08 The Regents Of The University Of California Array for detecting microbes
EP2446062A2 (fr) * 2009-06-26 2012-05-02 The Regents of the University of California Procédés et systèmes d'analyse phylogénétique
EP2446062A4 (fr) * 2009-06-26 2013-03-20 Univ California Procédés et systèmes d'analyse phylogénétique
US20140200149A1 (en) * 2012-03-06 2014-07-17 The Regents Of The University Of California Methods and compositions for identification of source of microbial contamination in a sample
US9725770B2 (en) * 2012-03-06 2017-08-08 The Regents Of The University Of California Methods and compositions for identification of source of microbial contamination in a sample
US10961593B2 (en) 2012-03-06 2021-03-30 The Regents Of The University Of California Methods and compositions for identification of source of microbial contamination in a sample

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AU1337099A (en) 1999-05-17

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