WO1999022023A2 - Method for identifying micro-organisms - Google Patents

Method for identifying micro-organisms 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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Prior art keywords
hybridization
microorganisms
substrate
seq
oligonucleotides
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PCT/EP1998/006863
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German (de)
French (fr)
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WO1999022023A3 (en
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Matthias Leiser
Bernd Epping
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Mira Diagnostica Gmbh
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Priority to AU13370/99A priority Critical patent/AU1337099A/en
Publication of WO1999022023A2 publication Critical patent/WO1999022023A2/en
Publication of WO1999022023A3 publication Critical patent/WO1999022023A3/en

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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
    • C12Q1/6813—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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Abstract

A method for identifying micro-organisms belonging to various taxes of micro-organisms, as specified in table 1, in a sample which can contain a plurality of various micro-organisms of said taxes, by means of nucleic acid hybridization techniques using oligo nucleotides with sequence ID. No. 1-62 as probes in order to obtain a hybridization result, whereby at least one hybridization result is obtained for each micro-organism to be identified.

Description

Verfahren zum Nachweis von Mikroorganismen Methods for the detection of microorganisms
Gegenstand der Erfindung ist ein Verfahren zum Nachweis von Mikroorganismen verschiedener Taxa in einer Probe, die eine Vielzahl von verschiedenen Mikroorganismen enthalten kann.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.
Die Identifikation von Mikroorganismen aus komplexen Proben (die mehrere unterschiedliche Keime im Gemisch enthalten) ist eine wichtige und schwierige Aufgabe, zum Beispiel bei Hygieneuntersuchungen und anderen Vorhaben.The identification of 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.
Die WO-A-97/41253 beschreibt ein Verfahren zum Nachweis von einem Mikroorganismus oder mehreren Mikroorganismen in einer Probe, die eine Vielzahl von verschiedenen Mikroorganismen enthält, mittels molekularbiologischer Techniken, wie Amplifikationsreaktionen, wobei mindestens eine Hybridisierungs- sonde (A), die konservierte Nucleinsäuresequenzen in dem oder den interessierenden Mikroorganismus(men) anzuzeigen in der Lage ist und mindestens eine Hybridisierungssonde (B), die weniger konservierte Nucleinsäuresequenzen in dem oder den interessierenden Mikroorganismus(men) anzuzeigen in der Lage ist, zu der Probe gegeben werden, mit der Maßgabe, daß pro interessierendem Mikroorganismus mindestens eine Hybridisierungssonde des Typs (A) und des Typs (B) vorhanden sein muß, sich die Probe in einem hybridisierungsfähigen Zustand befindet und durch ein entstehendes Hybridisierungsmuster eine Identifikation des oder der interessierenden Mikroorganismen erfolgt. Nachteilig an der geschilderten Methode ist, daß viele Bakterienarten aufgrund zu geringer Sequenzvariationen im Bereich der ribosomalen Gene, speziell der 16S rDNA, häufig Kreuzreaktionen mit den gewählten Oligonucleotid-Sequenzen zeigen und demzufolge nicht voneinander differenziert werden können.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 betrifft ein Verfahren zur simultanen Detektion und Identifikation und Differentiation von Eu-Bakterien unter Verwendung eines Hybridisationassays. Dabei werden im wesentlichen 16S-23S rR A Spacerbereiche amplifiziert und die erhaltenen Nucleinsäuren mit Sonden spezifischer Art hybridisiert. Der Nachteil dieser Methode beruht darin, daß der 16S-23S-Spacerbereich bei vielen Mikroorganismen kein funktioneller Abschnitt ist und deswegen keinem oder nur einem sehr geringen Selektionsdruck unterliegt. Als Folge lassen sich selbst innerhalb einer Art in den verschiedenen rDNA-Operonen Unterschiede in den Längen und Sequenzen der Spacerbereiche nachweisen, die häufig keinen Bezug zu phylogenetischen Zusammenhängen erkennen lassen (T. Hain: Molekulare Identifizierung von Streptomyzeten über Sequenzanalyse der Spacerbereiche ribosomaler RNA-Operone: Diplomarbeit Naturwiss. Fak. Der TU Carolo Wilhemina Braunschweig, 1995, 63 S.) und deshalb für diagnostische Fragestellungen ungeeignet erscheinen. Ein weiterer Nachteil besteht darin, daß die Spacersequenzen bisher nur bei wenigen Arten untersucht sind und es demzufolge für sie im Unterschied zu den 16S rDNA-Sequenzen noch keine ausreichenden Sequenzdatenbanken gibt. Dies bedeutet, daß bislang nicht genügend Sequenzinformationen vorliegen, um Nachweismethoden auf der Basis der Spacerregionen für die wichtigsten Bakterienarten zu entwickeln, selbst wenn der erstgenannte Nachteil beherrschbar wäre.WO-A-96/00298 relates to a method for the simultaneous detection and identification and differentiation of Eu bacteria using a hybridization assay. Essentially 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. As a result, 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. Hain: Molecular identification of streptomycetes via sequence analysis of the spacer regions of ribosomal RNA operons : Diplomarbeit Naturwiss. Fak. From TU Carolo Wilhemina Braunschweig, 1995, 63 p.) And therefore appear to be unsuitable for diagnostic questions. Another disadvantage is that the spacer sequences have so far only been investigated in a few species, and consequently, in contrast to the 16S rDNA sequences, there are still no sufficient sequence databases for them. This means that there is not yet enough sequence information available to develop detection methods based on the spacer regions for the most important types of bacteria, even if the first-mentioned disadvantage could be mastered.
EP-A-0 497 464 AI betrifft einen mikrobiologischen Schnellassay durch in situ Hybridisation in wäßriger Lösung. Dabei werden die Mikroorganismen zunächst mit einer wäßrigen Zusammensetzung in Kontakt gebracht, wodurch die Mikroorganismen zum einen fixiert und zum anderen die Zellwände für Oligonucleotide durchlässig gemacht werden. Danach werden markierte Nuclein- säuresonden zugegeben, die mit bestimmten Nucleotidsequenzen in der zellulären Nucleinsäure komplementär sind, so daß sich eine Hybridisierungsreaktion einstellt, gefolgt von einer Detektion der hybridisierten Nucleinsäure Dieses Verfahren betrifft eine In-situ-Hybridisierungsmethode. Die Erkenntnisse und Methoden sind auf in- vitro-Analysen, wie sie bei PCR- und Hybridisierungstests in Reaktionsgefäßen mit isolierter Ziel-Nucleinsäure üblich sind, nicht übertragbar. In-situ-Methoden haben den Nachteil, daß Probenvorbereitung und/oder Testdurchführung, insbesondere was den Detektionsteil betrifft, sehr komplizierte und teure Geräte erfordert.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. Thereafter, 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 betrifft ein Verfahren zur Charakterisierung eines unbekannten Organismus in einer Probe durch Bestimmung der Position eines Teils oder der gesamten konservierten DNA des betreffenden Organismus relativ zur Position von Restriktionsendonuclease-Spaltungsstellen in der DNA, wobei sich ein für einen bestimmten Mikroorganismus charakteristisches Muster einstellt. Nachteilig an diesem Verfahren ist, daß die Identifizierung von Organismen anhand ihrer DNA- Spaltmuster nach Abbau durch Restriktionsendonucleasen sehr aufwendig ist und die Reinkultur der zu analysierenden Organismen verlangt.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.
Das der Erfindung zugrundeliegende technische Problem besteht mithin darin, zunächst die genannten Nachteile des Standes der Technik zu vermeiden und darüber hinaus auch dem Anwender ein Verfahren an die Hand zu geben, daß es ihm nach Maßgabe seiner analytischen Probleme erlaubt, ein Assay- Verfahren zusammenzustellen, daß auf die spezifischen Bedürfhisse eines Analysenproblems zugeschnitten ist. Gelöst wird dieses Problem durch ein Verfahren mit den Merkmalen des Anspruchs 1. Die Unteransprüche betreffen bevorzugte Ausführungsformen des erfindungsgemäßen Verfahrens.The technical problem on which the invention is based is therefore first of all to avoid the disadvantages of the prior art mentioned and, moreover, to provide the user with a method which allows him to put together an assay method in accordance with his analytical problems. that is tailored to the specific needs of an analytical problem. This problem is solved by a method having the features of claim 1. The subclaims relate to preferred embodiments of the method according to the invention.
Überraschenderweise gewährleisten die erfindungsgemäßen Oligonucleotide mit den Seq. ID. Nr. 1 - 62 gemäß Sequenzprotokoll eine praxisgerechte Bakterienidentifikation.Surprisingly, the oligonucleotides according to the invention with the Seq. ID. No. 1 - 62 a practical bacterial identification according to the sequence listing.
Die variablen Regionen ribosomaler Gensequenzen sind häufig benutzt worden, um phylogenetische Zusammenhänge zwischen unterschiedlichen Arten herzustellen und DNA-Sonden bzw. PCR-Startoligonucleotide für diagnostische oder analytische Zwecke zu entwerfen und zu verwenden.The 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.
Allerdings hat sich hierbei mittlerweile in der Fachwelt die Anschauung durchgesetzt, daß die Sequenzen insbesondere der kleinen ribosomalen RNA (16S rDNA von Prokaryonten) wenig geeignet sind, um enger verwandte Bakterienarten geschweige denn -stamme hinreichend voneinander unterscheiden zu können. So schreiben z. B. Barry et al. (The 16s/23s ribosomal spacer region as a target for DNA probes to identify eubacteria, PCR Methods and Applications J_(1991) 51 - 56), daß zu wenig Sequenzvariationen zwischen den 16S rRNA-Genen von eng verwandten Mikroorganismen beobachtet werden. Cilia et al. (Sequence heterogeneties among 16S ribosomal RNA sequences, and their effect on phylogenetic analyses at the species level, Molec. Biol. Evol. 3_(1996) 451 - 461) stellen fest, daß rRNA- Sequenzen der kleinen Untereinheit nicht adäquat geeignet sind, um phylogenetische Beziehungen zwischen eng verwandten Arten, geschweige denn verschiedenen Stämmen innerhalb einer Art zu analysieren. Sie zitieren hierzu weitere Autoren (Ash et al., Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA sequences, Lett. Appl. Microbiol. 13 (1991) 202 - 206; Rössler et al., 1991; Ruimy et al., 1994). Ähnlich äußern sich Berthier und Ehrlich (Rapid species identification with two groups of closely related lactobacilli using PCR primers that target the 16S/23S rRNA spacer region. Fems Microbiol. Letters 161 (1998) 97 - 106). rRNA-Sonden eng verwandter Arten können aufgrund der hohen Ähnlichkeit der rRNA-Sequenzen nicht verwendet werden. Sie zitieren hierzu weiterhin Fox et al. (How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identify, Int. J. Bacteriol. 42 (1992) 166 - 170), Schleifer et al. (Application of molecular methods for the classification and identification of lactic acid bacteria, Int. Dairy J. 5 (1995) 1081 - 1094) und Curk et al. (Lactobacillus paraplantarum sp. Nov., A new species related to Lactobacillus plantarum, Int. J. Syst. Bacteriol. 46 (1996) 595 - 598.In the meantime, however, the view has prevailed in the professional world that the sequences, in particular of the small ribosomal RNA (16S rDNA from prokaryotes), are not very suitable for being able to sufficiently differentiate between closely related types of bacteria, let alone strains. So write z. B. Barry et al. (The 16s / 23s ribosomal spacer region as a target for DNA probes to identify eubacteria, PCR Methods and Applications J_ (1991) 51 - 56) that too few sequence variations between the 16S rRNA genes are observed by closely related microorganisms. Cilia et al. (Sequence heterogeneties among 16S ribosomal RNA sequences, and their effect on phylogenetic analyzes at the species level, Molec. Biol. Evol. 3_ (1996) 451-461) state that rRNA sequences of the small subunit are not adequately suited to Analyze phylogenetic relationships between closely related species, let alone different strains within a species. They cite further authors (Ash et al., Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA sequences, Lett. Appl. Microbiol. 13 (1991) 202-206; Rössler et al., 1991 ; Ruimy et al., 1994). Berthier and Ehrlich (Rapid species identification with two groups of closely related lactobacilli using PCR primers that target the 16S / 23S rRNA spacer region. Fems Microbiol. Letters 161 (1998) 97-106). rRNA probes from closely related species cannot be used due to the high similarity of the rRNA sequences. You continue to quote Fox et al. (How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identify, Int. J. Bacteriol. 42 (1992) 166-170), Schleifer et al. (Application of molecular methods for the classification and identification of lactic acid bacteria, Int. Dairy J. 5 (1995) 1081-1094) and Curk et al. (Lactobacillus paraplantarum sp. Nov., A new species related to Lactobacillus plantarum, Int. J. Syst. Bacteriol. 46 (1996) 595-598.
Das erfindungsgemäße Verfahren erlaubt auch eine simultane Erfassung und simultanen Nachweis von Mikroorganismen verschiedener Taxa in einer Probe, die eine Vielzahl von verschiedenen Mikroorganismen enthalten kann. Unter dem Begriff "Taxon" werden Familien, Gattungen, Arten und Unterarten von Mikroorganismen verstanden.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. The term "taxon" means families, genera, species and subspecies of microorganisms.
Basis für das erfindungsgemäße Verfahren ist die Anwendung von Nucleinsäurehybridisierungstechniken. Dabei werden Hybridisierungssonden eingesetzt, die mit DNA oder RNA, die indikativ für Mikroorganismen sind und aus den nachzuweisenden Organismen stammen, in Wechselwirkung treten. Ist die Konzentration an nachzuweisender Nucleinsäure zu gering, können gegebenenfalls Amplifikationstechniken zur Erhöhung der Konzentration eingesetzt werden. Durch Hybridisierung der Sonden mit der DNA oder RNA wird ein Hybridisierungsergebnis erhalten. Erfindungsgemäß ist dabei wesentlich, daß für jeden nachzuweisenden Mikroorganismus zunächst mindestens ein Hybridisierungsergebnis erhalten wird. Dies kann insbesondere durch das in der WO-A-97/41253 beschriebene Verfahren erfolgen. Auf den Gegenstand der WO-A-97/41253 wird ausdrücklich Bezug genommen.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.
Als Sonden werden erfindungsgemäß Oligonucleotide eingesetzt, die einer der im Sequenzprotokoll mit den Seq. Id. Nr. 1 - 62 ausgewiesenen Sequenzen besitzt. Dabei kann erfindungsgemäß sowohl das gesamte Ensemble der Oligonucleotide mit den Nr. 1 - 62 eingesetzt werden oder aber der Anwender sucht die zu seinem Nachweisproblem zugehörigen Oligonucleotide mit der Maßgabe der Zuordnung gemäß Tabelle 1 aus. Erfindungswesentlich ist, daß die Tabelle 1 angibt, welche der genannten Oligonucleotidsequenzen indikativ für das betreffende Taxon ist.According to the invention, 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.
Figure imgf000008_0001
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000009_0001
Acinetobacter anitra (DSM 30008), Acinetobacter baumannii (DSM 30007), Acinetobacter haemolyticusAcinetobacter 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 SpeziesAcinetobacter 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 bedeutet "positiv" Die Tabelle 2 stellt die Konkordanz zwischen den Oligonucleotiden und deren Bezeichnung gemäß Tabelle 1 her.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.
Als Starteroligonucleotid wurde 10-30f mit der SequenzThe starting oligonucleotide was 10-30f with the sequence
GAG TTT GAT CCT GGC TCA G verwendet (Seq. Id. Nr. 62).GAG TTT GAT CCT GGC TCA G used (Seq. Id. No. 62).
Tabelle 2Table 2
ID-Nr. Abkürzung Sequenz 5'-3'ID no. Abbreviation sequence 5'-3 '
1. Ac.anlr CAC TAT CTC TAG GTA TTA ACT AAA GT1. Ac.anlr CAC TAT CTC TAG GTA TTA ACT AAA GT
2. Ae.calr AGG TATTAA CTT CAG TAG CC2. Ae.calr AGG TATTAA CTT CAG TAG CC
3. Ac.xxlr CGA GTAACG TCC ACT ATC TG3. Ac.xxlr CGA GTAACG TCC ACT ATC TG
4. Ae.calr CCA GCA GAT ATT AGC TAC TG4. Ae.calr CCA GCA GAT ATT AGC TAC TG
5. Ae.hy lr TTG ATA CGTATT AGG CAT CA5. Ae.hy lr TTG ATA CGTATT AGG CAT CA
6. Ae.hy 2r GTT GATACG TAT TAG GCA TCA6. Ae.hy 2r GTT GATACG TAT TAG GCA TCA
7. Ae.salr TTG ACA CGTATT AGG CGC7. Ae.salr TTG ACA CGTATT AGG CGC
8. Ae.sclr TGG CAG GTA TTAACC ACC A8. Ae.sclr TGG CAG GTA TTAACC ACC A
9. Al.fa2r TCT CGTATTAGG AGATAC CTT9. Al.fa2r TCT CGTATTAGG AGATAC CTT
10. Al.xxlr TAC TGG GCA CGTTCC GATAT10. Al.xxlr TAC TGG GCA CGTTCC GATAT
11. Al.xx2r ATATCG GCC GCT CCAATA GT11. Al.xx2r ATATCG GCC GCT CCAATA GT
12. Ba.ce gr2r TAC CGT CAA GGT GCC AGC T12. Ba.ce gr2r TAC CGT CAA GGT GCC AGC T
13. Ba.ce/stplr CCATGC GGT TCA AAA TGT T13. Ba.ce / stplr CCATGC GGT TCA AAA TGT T
14. Ba.ce grlr CCAGCT TAT TCAACTAGC14. Ba.ce grlr CCAGCT TAT TCAACTAGC
15. Ba.la grlr CGG AAA CCC TCC AAC A15. Ba.la grlr CGG AAA CCC TCC AAC A
16. Ba.su2r ACC GCC CTATTC GAA CGG T16. Ba.su2r ACC GCC CTATTC GAA CGG T
17. Br.th2r AGC GCG GGT CCA TCT CAC17. Br.th2r AGC GCG GGT CCA TCT CAC
18. Br.th3r CAT CTTATG ATG TTC AGC ACA18. Br.th3r CAT CTTATG ATG TTC AGC ACA
19. Ca.dilr CC ATG CGG TCA CTT GAAAT19. Ca.dilr CC ATG CGG TCA CTT GAAAT
20. Ca.jelr AG TGT CAT CCT CCA20. Ca.jelr AG TGT CAT CCT CCA
21 Ca.je2r ATT CTT CCC TAA GAAAAG GAG21 Ca.je2r ATT CTT CCC TAA GAAAAG GAG
22. Ca.je3r CGT CAG AAT TCT TCC CTA AG22. Approx. Each CGT CAG AAT TCT TCC CTA AG
23. Ca.pi/galr TCA TGC GAT TCC TGA AAC23. Ca.pi / galr TCA TGC GAT TCC TGA AAC
24. Ci.fr2r GTAAC GTC AAT GGC TGA GGT24. Ci.fr2r GTAAC GTC AAT GGC TGA GGT
25. Ci.fr3r TT CTC TGGATG TCA AGA GT25. Ci.fr3r TT CTC TGGATG TCA AGA GT
26. Ci.fr5r CC AAG GCATCT CTG CCA AG26. Ci.fr5r CC AAG GCATCT CTG CCA AG
27. Cl.pelr CC TTT GGT TGAATG ATG27. Cl.pelr CC TTT GGT TGAATG ATG
28. Ed.talr CC CGTATC TCT ACA GGA28. Ed.talr CC CGTATC TCT ACA GGA
29. En.ae2r GAG TAA CGT CAA TCG CCA AG ID-Nr. Abkürzung Sequenz 5'-3' 29. En.ae2r GAG TAA CGT CAA TCG CCA AG ID no. Abbreviation sequence 5'-3 '
30. En.cBr AG CCG TTA CCC CAC CTA CT30. En.cBr AG CCG TTA CCC CAC CTA CT
31. Es.co2r GCA AAG GTA TTA ACT TTA CTC31. Es.co2r GCA AAG GTA TTA ACT TTA CTC
32. Es.co3r GTA ACG TCA ATG AGC AAA GG32. Es.co3r GTA ACG TCA ATG AGC AAA GG
33. Fl.brlr TA CGC ATG CCT ATC CTA CT33. Fl.brlr TA CGC ATG CCT ATC CTA CT
34. Fl.br2r TG GTA CCT TCA GCT ACT TA34. Fl.br2r TG GTA CCT TCA GCT ACT TA
35. Fl.odlr CCA TGG AGC ATT AAT CCG AA35. Fl.odlr CCA TGG AGC ATT AAT CCG AA
36. Gamma 1 AAG GTC CCC CTC TTT GGT36. Gamma 1 AAG GTC CCC CTC TTT GGT
37. Ha.al2r GT AAC GTC AAT CAC TGT GG37. Ha.al2r GT AAC GTC AAT CAC TGT GG
38. Kl.ox3r GGT AAC GTC AAT GAA TAA GGT38. Kl.ox3r GGT AAC GTC AAT GAA TAA GGT
39. La.palr CAA CAG TTA CTC TGC CGA CCA39. La.palr CAA CAG TTA CTC TGC CGA CCA
40. La.pa2r TTA CGC CAT CTT TCA GCC A40. La.pa2r TTA CGC CAT CTT TCA GCC A
41. Li.mo2 r CAA GCA GTT ACT CTT AT41. Li.mo2r CAA GCA GTT ACT CTT AT
42. Mi.la2r AT TTC TGG CCC GTT CTC GT42. Mi.la2r AT TTC TGG CCC GTT CTC GT
43. Mi.xxlr ATT TCT GGC CCG TTC TCG43. Mi.xxlr ATT TCT GGC CCG TTC TCG
44. Mo.bolr CTA TCT CTA GCG AAT TCT TGG44. Mo.bolr CTA TCT CTA GCG AAT TCT TGG
45. Mo.ca2r GGT AAC GTC AGG GCT TAT G45. Mo.ca2r GGT AAC GTC AGG GCT TAT G
46. Pe.da/palr TGG ATA CCG TCA CTG CAT GAG46. Pe.da / palr TGG ATA CCG TCA CTG CAT GAG
47. Pl.shlr CCA CTA GGT ATT AAC TAG TGA47. Pl.shlr CCA CTA GGT ATT AAC TAG TGA
48. Pr.vu3r AAC CCC TGC TTT GGT CCG TA48. Pr.vu3r AAC CCC TGC TTT GGT CCG TA
49. Ps.aelr CCG TAC TCT AGC TCA GT49. Ps.aelr CCG TAC TCT AGC TCA GT
50. Ra.pilr GGT ATTAAC CAG AGC CAT50th Ra.pilr GGT ATTAAC CAG AGC CAT
51. Ra.pi2r TAG CCG TGC AGT CAC CA51st Ra.pi2r TAG CCG TGC AGT CAC CA
52. Sa.ty2 r CTG CGG TTA TTA ACC ACA ACA52. Sa.ty2r CTG CGG TTA TTA ACC ACA ACA
53. Sa.xx5r ACC AAT CCA TCT CTG GAT TC53. Sa.xx5r ACC AAT CCA TCT CTG GAT TC
54. Se.malr ATG AGC GTA TTA AGC TCA CCA54. Se.malr ATG AGC GTA TTA AGC TCA CCA
55. St.au/halr GGC TCTATC TCT AGA GTT G55. St.au/halr GGC TCTATC TCT AGA GTT G
56. St.au2r pre GTG CAC AGT TAC TTA CAC ATA56. St.au2r pre GTG CAC AGT TAC TTA CAC ATA
57. Str.aglr ATT TTC CAC TCC TAC CAA C57.str.aglr ATT TTC CAC TCC TAC CAA C
58. Str.ag3r CCG TTT CCA AAG CGT ACAAT58th Str.ag3r CCG TTT CCA AAG CGT ACAAT
59. Vi.vulr GCT AAC GTC AAA TGA TAG TGC59th Vi.vulr GCT AAC GTC AAA TGA TAG TGC
60. Vi.vu2r GCT AAC GTC AAA TGA TGC CGC60. Vi.vu2r GCT AAC GTC AAA TGA TGC CGC
61. Ye.enlr AAC AAC GTA TTA AGT TATTGG61. Ye.enlr AAC AAC GTA TTA AGT TATTGG
62. 10-30f GAG TTT GAT CCT GGC TCA G62. 10-30f GAG TTT GAT CCT GGC TCA G
Vorzugsweise wird erfindungsgemäß eine weitere Hybridisierung mittels mindestens einer Hybridisierungssonde durchgeführt, die von der ersten Hybridisierungssonde unterschiedlich ist, aber auch eine der Seq. ID Nr. 1 - 61 aufweist. Dies führt dann entweder zum Auftreten oder zum Ausbleiben von Kreuzreaktionen. Diese Information kann dann zur eindeutigen Identifikation der Mikroorganismen oder eines Mikroorganismenensembles in einer Probe heranzogen werden.According to the invention, 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.
Vorteilhaft am erfindungsgemäßen Verfahren ist mithin die Möglichkeit, in eindeutiger Weise bestimmte Mikroorganismen allein oder ein Ensemble von Mikroorganismen simultan zu bestimmen. Die Auswertung der Hybridisierungsergebnisse kann dann beispielsweise durch eine zweidimensionale Auftragung der Hybridisierungsergebnisse erfolgen. Es ergibt sich dabei ein bestimmtes Hybridisierungsmuster, welches indikativ für ein bestimmtes Mikroorganismenensemble ist. Solche Muster können z.B. in Datenbanken abgelegt werden. Insbesondere bei Reihenuntersuchungen können solche Muster abgefragt werden und zur schnellen und sicheren Identifizierung eingesetzt werden. Das erfindungsgemäße Verfahren eignet sich mithin insbesondere für automatisierbare Untersuchungen.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.
Vorzugsweise erfolgt eine Detektion der Kreuzreaktion räumlich und/oder zeitlich oder durch verschiedene Markierungen der Sonden.The cross-reaction is preferably detected spatially and / or temporally or by means of different markings on the probes.
Vorzugsweise kann die räumliche Anordnung der Hybridisierungsergebnisse auf einem Substrat erfolgen. Als Substrate kommen beliebige, in der Molekularbiologie gebräuchliche Trägersysteme in Betracht, wie beispielsweise Mikrotite latten, Blotting-Papiere, Spezialmembranen oder DNA-Chips.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.
Eine zeitliche Auflösung der Kreuzreaktion bietet sich bei Durchflußverfahren an. Es werden der Probe sequentiell Hybridisierungssonden zugesetzt und deren Wechselwirkung mit in der Probe enthaltener Nucleinsäure untersucht.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.
Alternativ kann auch bei einer statischen Analyse durch Wahl geeigneter Markierungsreagenzien eine zeitliche Aufnahme der Hybridisierungsergebnisse in Mustern erfolgen. Beispielsweise ist dies möglich durch Verwendung von Farbstoffen unterschiedlicher spektraler Charakteristika oder von Fluoreszenzmarkem mit kurzer, unterschiedlicher oder zeitversetzter Lebensdauer. Auch die in zeitlicher Abhängigkeit aufgenommenen Hybridisierungsmuster können in Datenbanken abgelegt werden und dann bei Abgleich mit einer aktuellen Probe als Hinweis für den mikrobiologischen Zustand dieser Probe dienen.Alternatively, in the case of a static analysis, by selecting suitable labeling reagents, 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.
Gegenstand der vorliegenden Erfindung sind mithin auch Oligonucleotide mit denen in Seq. Id. Nr. 1 - 61 wiedergegebenen Sequenzen.The present invention therefore also relates to oligonucleotides with those in Seq. Id. Nos. 1-61 reproduced sequences.
Die erfindungsgemaßen Oligonucleotide können insbesondere in Form von Kits zusammen mit Hilfsmitteln zur Durchführung des erfindungsgemäßen Verfahrens angeboten werden. In vorteilhafter Weise sind dabei die Oligonucleotide auf einem Substrat angeordnet. Die Anordnung kann insbesondere in festen Zuordnungen erfolgen, so daß bei positiver Hybridisierung eine Detektion auf einem Substrat jeweils an einer standardisierten gleichen Stelle erscheint, was eine Automatisierung der Auswertung erleichtert.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.
Das erfindungsgemäße Verfahren wird anhand der folgenden Beispiele näher erläutert.The process according to the invention is explained in more detail with the aid of the following examples.
Beispiel 1example 1
Voranreicherung und Extraktion der MikroorganismenPre-enrichment and extraction of the microorganisms
Zur Voranreicherung werden 100 ml des Probenmaterials mit 300 ml nicht selektivem Flüssigmedium (CASO-Bouillon), versetzt und für 4 - 18 h im Brutschrank bei 28 - 37°C (abhängig von den Zielorganismen) bebrütet. Anschließend erfolgt die Entnahme von 1,5 ml dieser Flüssiganreicherung für die nachfolgende Extraktion der DNA. Diese Lösung wird für 2 min bei 8000 g zentrifugiert, der entstandene Überstand verworfen und das Sediment (Pellet) in 100 μl Puffer 1 (2 mg/ml Lysozym, 20 μg/ml Lysostaphin, 100 mM Tris/HCl pH 7,2 - 7,4, 2 mM CaCl2, 4 % Saccharose-Lösung, Proteinase K-Lösung (20 mg/ml H2O)) resuspendiert. Danach erfolgt die Zugabe von 20 μl RNase A-Lösung (20 mg/ml in Natriumacetatpuffer, pH 5,2) und Vermischung der Suspension auf dem Schüttler. Im Anschluß daran wird das Gemisch für 10 min bei 60°C im Schüttelwasserbad inkubiert. Anschließend werden dieser Lösung 100 μl Puffer 2 (10% SDS-Lösung, 1,5 mM EDTA) zugegeben und nochmals für 10 min bei 60°C im Schüttelwasserbad inkubiert. Die Reinigung der DNA erfolgt mittels DNA- Reinigungssäulen (QIAamp von QIAGEN). Hierzu wird die Lösung mit 200 μl Bindungspuffer (AL-Puffer QIAGEN) und 200 μl absolutem Alkohol versetzt und auf dem Schüttler gut durchmischt. Das gesamte Volumen wird anschließend auf eine Reinigungssäule (in einem Leertube fixiert) gegeben und für 1min bei 8000 g zentrifugiert. Das Zentrifugat wird verworfen, die Reinigungssäule in ein neues Leertube gegeben und mit 500 μl Waschpuffer (AW-Puffer von QIAGEN) beschickt. Es folgt eine erneute Zentrifugati on für 1min bei 8000 g und Verwerfen des entstandenen Zentrifugates. Danach werden 200 μl H2O (auf 60°C vorgewärmt) zugegeben und für 1min bei 8000 g zentrifugiert. Die auf diesem Wege eluierte DNA kann nun für die nachfolgende Identifizierung mittels PCR herangezogen werden.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. This solution is at 8000 g for 2 min centrifuged, the resulting supernatant discarded and the sediment (pellet) in 100 μl buffer 1 (2 mg / ml lysozyme, 20 μg / 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 μl 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. in a shaking water bath. Then 100 μl of buffer 2 (10% SDS solution, 1.5 mM EDTA) are added to this solution and incubated again for 10 min at 60 ° C. in a shaking water bath. The DNA is cleaned using DNA cleaning columns (QIAamp from QIAGEN). For this purpose, the solution is mixed with 200 μl binding buffer (AL buffer QIAGEN) and 200 μl 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 μl washing buffer (AW buffer from QIAGEN). A new centrifugation follows for 1 min at 8000 g and the resulting centrifugate is discarded. Then 200 μl H 2 O (preheated to 60 ° C.) are added and centrifuged for 1 min at 8000 g. The DNA eluted in this way can now be used for the subsequent identification by means of PCR.
Identifizierung der Mikroorganismen mittels PCRIdentification of the microorganisms using PCR
Zur Durchführung der PCR werden folgende Reaktionskomponenten miteinander vermischt: 10 x PCR-Puffer (0,1 % Tween, 660 mM Tris/HCl pH 8,8, 166 mM (NH4)2SO4 5,0 μlThe following reaction components are mixed with one another to carry out the PCR: 10 x PCR buffer (0.1% Tween, 660 mM Tris / HCl pH 8.8, 166 mM (NH 4 ) 2 SO 4 5.0 ul
MgCl2 (25 mM) 5,0 μl dNTP's (je 2 mM) 5,0 μlMgCl 2 (25 mM) 5.0 ul dNTP's (2 mM each) 5.0 ul
Taq-Polymerase (5 U/μl) 0,2 μlTaq polymerase (5 U / µl) 0.2 µl
DNA-Template (0,2 ng/μl) 5,0 μlDNA template (0.2 ng / µl) 5.0 µl
Primer A (30 pmol) 1,0 μlPrimer A (30 pmol) 1.0 ul
Primer B (30 pmol) 1 ,0 μlPrimer B (30 pmol) 1.0 ul
H2O 27,8 μlH 2 O 27.8 ul
Die Amplifikation der Ziel-DNA (Target-DNA) erfolgt in einem Thermocycler (GeneAmp PCR System 9700 von Perkin Eimer) mit vorzugsweise beheiztem Deckel. Hierbei wird die DNA zuerst für 5 min bei 94°C denaturiert und anschließend für 30 Zyklen unter folgenden Bedingungen amplifiziert: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:
30 see 94°C Zyklus-Denaturierung30 see 94 ° C cycle denaturation
15 see 56°C Primer-Hybridisierung („Annealing,,)15 see 56 ° C primer hybridization ("Annealing")
20 see 72°C Elongation („Extension,,)20 see 72 ° C elongation ("Extension")
Zum Abschluß des PCR-Programmes erfolgt eine nochmalige Elongation bei 72°C für 1min.At the end of the PCR program, another elongation takes place at 72 ° C for 1min.
Die Auswahl der Starteroligonucleotide (Primer) bestimmt letztendlich die Taxonspezifität der PCR- Amplifikation und ermöglicht somit die Identifizierung der in der Probe vorhandenen Bakterien. Hierbei kann der Primer A sowohl zur taxonspezifischen Hybridisierung an einen variablen Bereich der Ziel-DNA, als auch zur breitbandspezifischen Hybridisierung an einen in allen Bakterien homologen Sequenzbereich dienen. Hingegen hybridisiert Primer B an einen artspezifischen Sequenzbereich der Bakterien-DNA und ist somit der entscheidende Faktor zur Bestimmung der Spezies/Gattung oder Gruppe. Für jeden nachzuweisenden Organismus werden die jeweils benötigten Starteroligonucleotide A und B benötigt.The selection of the starter oligonucleotides (primers) ultimately determines the taxon specificity of the PCR amplification and thus enables the identification of the bacteria present in the sample. Here, 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. In contrast, 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.
Beispiel 2: MilchExample 2: Milk
Bei den zum Nachweis in Milch relevanten Bakterien handelt es sich in erster Linie um folgende Gattungen/Spezies:The bacteria relevant for detection in milk are primarily the following genera / species:
E. coliE. coli
Campylobacter Spezies Listeria Spezies Salmonella speziesCampylobacter species Listeria species Salmonella species
Zur Identifizierung der oben genannten Bakterien werden die nachfolgend aufgeführten DNA- Starteroligonucleotide herangezogen:The DNA starter oligonucleotides listed below are used to identify the bacteria mentioned above:
Gattung/Spezies Starteroligonucleotid A Starteroligonucleotid BGenus / Species Starter oligonucleotide A Starter oligonucleotide B
E.coli 10-30f (Seq. ID. Nr. 62) Es.co3r (ID-Nr. 32)E.coli 10-30f (Seq. ID No. 62) Es.co3r (ID No. 32)
Campylobacter spezies 10-30f Ca.jelr (ID-Nr. 20) Listeria spezies 10-30f Li.mo2r (ID-Nr. 41) Salmonella spezies 10-30f Sa.xx5r (ID-Nr. 53) 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)
Beispiel 3: WasserExample 3: Water
Bei den zum Nachweis in Wasser relevanten Bakterien handelt es sich in ersterThe bacteria relevant for detection in water are first
Linie um folgende Gattungen/Spezies:Line around the following genera / species:
E.coliE. coli
Enterobacter aerogenesEnterobacter aerogenes
Klebsiella oxytocaKlebsiella oxytoca
Pseudomonas aeruginosaPseudomonas aeruginosa
Enterobakterien6) Enterobacteria 6)
Zur Identifizierung der oben genannten Bakterien werden die nachfolgend aufgeführten DNA-Starteroligonucleotide herangezogen:The DNA starter oligonucleotides listed below are used to identify the bacteria mentioned above:
Gattung/Spezies Starteroligonucleotid A Starteroligonucleotid BGenus / Species Starter oligonucleotide A Starter oligonucleotide B
E.coli 10-30f Es.co3r (ID-Nr. 32)E.coli 10-30f Es.co3r (ID No. 32)
Enterobacter aerogenes 10-30f En.ae2r (ID-Nr. 29)Enterobacter aerogenes 10-30f En.ae2r (ID No. 29)
Klebsiella oxytoca 10-30f Kl.ox3r (ID-Nr. 38)Klebsiella oxytoca 10-30f Kl.ox3r (ID No. 38)
Pseudomonas aeruginosa 10-30f Ps.aelr (ID-Nr. 49)Pseudomonas aeruginosa 10-30f Ps.aelr (ID No. 49)
Enterobakterien6) 10-30f Gammair (ID-Nr.36)Enterobacteria 6) 10-30f Gammair (ID-No.36)
6) 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 Beispiel 4: Für unterschiedliche in der Praxis vorkommende Proben wurden artifizielle Gemische im Experiment untersucht (Tabelle 2) 6) 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)
Häufige Fragestellungen in der Praxis erfordern vielfach eine Identifizierung von ganz bestimmten Keimen oder eines reduzierten Artenspektrums. Dies liegt in den ganz unterschiedlichen Wachstumsanforderungen der Bakterien begründet, das heißt, daß nicht alle Bakterienarten gleichsam in unterschiedlichen Proben wachsen oder leben können. Hieraus ergibt sich für Untersuchungen an Proben häufig die Bestimmung ganz bestimmter Bakterienarten, -gattungen, oder -gruppen (Beispiele 2 und 3). In Tabelle 1 sind Identifizierungen von Organismen dargestellt, die in ganz unterschiedlichen artifiziellen Gemischen hinsichtlich dieser Prämisse geprüft und einwandfrei bestimmt werden konnten.Frequent questions in practice often require the identification of very specific germs or a reduced spectrum of species. This is due to the very different growth requirements of the bacteria, which means that not all types of bacteria can grow or live in different samples, as it were. For tests on samples, this often results in the determination of certain types, types or groups of bacteria (Examples 2 and 3). Table 1 shows the identifications of organisms that could be tested in various artificial mixtures with regard to this premise and determined without problems.
Zur Identifizierung werden die Amplifikate der PCR-Reaktion auf ein 1 % Agarosegel aufgetragen und bei 5 - 6 V/cm Elektrodenabstand im elektrischen Feld durch Horizontalgelelektrophorese aufgetrennt. Anschließend wird das Agarosegel für 10min in einer 0,5 μg/ml Ethidiumbromid/TAE-Puffer-Lösung gefärbt und unter einem UV-Transilluminator bei 254 nm photodokumentiert.For identification, 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 μg / ml ethidium bromide / TAE buffer solution and photo-documented at 254 nm under a UV transilluminator.
Die entwickelten Starteroligonucleotide weisen die genannten Bakterien auch in Mischproben hochspezifisch nach. Sofern eine Probe eine oder mehrere der gesuchten Bakterien enthält, wird dies durch eine Bande mit definierter Größe auf dem Elektrophoresegel sichtbar. 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.

Claims

Ansprüche Expectations
1. Verfahren zum Nachweis von in Tabelle 1 angegebenen Mikroorganismen verschiedener Taxa in einer Probe, die eine Vielzahl von verschiedenen Mikroorganismen dieser Taxa enthalten kann, mittels Nucleinsäurehybri- disierungstechniken bei Verwendung von Oligonucleotiden mit der Seq. ID. No 1 bis 62 als Sonden, unter Erhalt eines Hybridisierungsergebnisses, wobei für jeden nachzuweisenden Mikroorganismus mindestens ein Hybridisierungsergebnis erhalten wird.1. Method for the detection of microorganisms of different taxa given in Table 1 in a sample, which may contain a large number of different microorganisms of these taxa, by means of nucleic acid hybridization techniques using oligonucleotides with the Seq. ID. No 1 to 62 as probes, with a hybridization result, whereby at least one hybridization result is obtained for each microorganism to be detected.
2. Verfahren nach Anspruch 1, wobei mittels einer Kreuzreaktion durch Zugabe mindestens einer zweiten von der ersten Sonde verschiedenen Hybridisierungssonde, die ausgewählt ist aus der Gruppe der Oligonucleotide mit den Seq. ID No 1 bis 62 nach Maßgabe der in Tabelle 1 erfolgten Zuordnung, eine eindeutige Identifizierung des nachzuweisenden Mikroorganismus oder Gruppe von Mikroorganismen erfolgt.2. The method according to claim 1, wherein by means of a cross reaction by adding at least one second hybridization probe different from the first probe, which is selected from the group of the oligonucleotides with the Seq. ID No 1 to 62 in accordance with the assignment made in Table 1, the microorganism or group of microorganisms to be detected is clearly identified.
3. Verfahren nach Anspruch 2, wobei eine Detektion der Kreuzreaktion räumlich und/oder zeitlich aufgelöst erfolgt.3. The method according to claim 2, wherein a detection of the cross reaction is spatially and / or temporally resolved.
4. Verfahren nach Anspruch 3, dadurch gekennzeichnet, daß die Detektion durch Verwendung unterschiedlicher Markierungen der Hybridisierungs- sonden erfolgt.4. The method according to claim 3, characterized in that the detection is carried out by using different labels of the hybridization probes.
5. Verfahren nach mindestens einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, daß die Hybridisierungsergebnisse auf einem Substrat angeordnet sind. 5. The method according to at least one of claims 1 to 4, characterized in that the hybridization results are arranged on a substrate.
6. Verfahren nach Anspruch 5, wobei die auf dem Substrat angeordneten Hybridisierungsergebnisse ein für die nachzuweisenden Mikroorganismen charakteristisches Muster ergeben.6. The method according to claim 5, wherein the hybridization results arranged on the substrate result in a pattern characteristic of the microorganisms to be detected.
7. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß das Hybridisierungsergebnis in Abhängigkeit von der Zeit aufgenommen wird.7. The method according to claim 1, characterized in that the hybridization result is recorded as a function of time.
8. Verfahren nach Anspruch 7, dadurch gekennzeichnet, daß das zeitlich aufgenommene Hybridisierungsmuster spezifisch für die nachzuweisenden Mikroorganismen oder ein Ensemble von Mikroorganismen ist.8. The method according to claim 7, characterized in that the temporally recorded hybridization pattern is specific for the microorganisms to be detected or an ensemble of microorganisms.
9. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß ein Hybridisierungsmuster durch Aufnahme von Hybridisierungsergebnissen erstellt wird, die durch Sonden mit unterschiedlicher Markierung erstellt werden.9. The method according to claim 1, characterized in that a hybridization pattern is created by recording hybridization results, which are created by probes with different labels.
10. Verfahren nach Anspruch 5, dadurch gekennzeichnet, daß das Substrat ein DNA-Chip ist.10. The method according to claim 5, characterized in that the substrate is a DNA chip.
11. Substrat mit mindestens einer Art von Oligonucleotiden gemäß Seq. ID. No 1 - 61.11. Substrate with at least one type of oligonucleotide according to Seq. ID. No 1-61.
12. Substrat nach Anspruch 11, wobei das Substrat als DNA-Chip ausgebildet ist. 12. The substrate of claim 11, wherein the substrate is designed as a DNA chip.
13. Kit enthaltend mindestens ein Oligonucleotid mit der Seq. Id. No 1 - 62, ggf. auf mindestens einem Substrat nach einem der Ansprüche 11 oder 12 sowie Hilfsmittel zu Durchführung des Verfahrens nach einem der Ansprüche 1 bis 10.13. Kit containing at least one oligonucleotide with the Seq. Id. No 1-62, possibly on at least one substrate according to one of claims 11 or 12 and aids for carrying out the method according to one of claims 1 to 10.
14. Oligonucleotide mit den in Seq. Id. Nr. 1 - 61 wiedergebenen Sequenzen. 14. Oligonucleotides with the in Seq. Id. No. 1 - 61 reproduced sequences.
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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
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