EP1252174A2 - Oligonucleotides permettant l'amplification specifique et la detection specifique de genes d'arnr 16s de bacteries - Google Patents

Oligonucleotides permettant l'amplification specifique et la detection specifique de genes d'arnr 16s de bacteries

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Publication number
EP1252174A2
EP1252174A2 EP01913774A EP01913774A EP1252174A2 EP 1252174 A2 EP1252174 A2 EP 1252174A2 EP 01913774 A EP01913774 A EP 01913774A EP 01913774 A EP01913774 A EP 01913774A EP 1252174 A2 EP1252174 A2 EP 1252174A2
Authority
EP
European Patent Office
Prior art keywords
oligonucleotides
dna
pcr
oligonucleotide
rna
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP01913774A
Other languages
German (de)
English (en)
Inventor
Hans-Jürgen BACH
Michael Schloter
Jean-Charles Munch
Jitka Tomanova
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Original Assignee
Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt 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 Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH filed Critical Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Publication of EP1252174A2 publication Critical patent/EP1252174A2/fr
Withdrawn legal-status Critical Current

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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
    • C12Q1/6876—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
    • C12Q2561/00—Nucleic acid detection characterised by assay method
    • C12Q2561/101—Taqman

Definitions

  • the invention relates to specific oligonucleotides
  • bacteria play an important role as function carriers in natural ecosystems
  • diseases are caused by a large number of microorganisms.
  • limit values were set for many natural products (food, drinking water, etc.) as to how many bacteria may be in a sample in order to classify the sample as hygienically harmless.
  • the 16S rRNA is a bacterial-specific ribosomal component and has therefore been used in the past for methods that should be used to demonstrate the presence of bacteria in sample materials.
  • the oligonucleotides previously used as primers and probes differ from the oligonucleotides disclosed according to the invention.
  • the oligonucleotides described here can be used particularly advantageously in the context of a TaqMan PCR.
  • Several papers have already been published, including the TaqMan PCR for quantitative and qualitative Detection of bacteria was used.
  • One example is Kimura et al, 1999, Journal of Food Protection. 62: 329- 335.
  • probes and primers were presented in these 'works that were suitable for quantitative and qualitative detection of specific organisms and groups of organisms.
  • oligonucleotides presented here are suitable in the form of an oligonucleotide set (forward primer, reverse primer and probe) for quantitative bacterial detection and, with regard to annealing temperatures, length of the generated amplicon and the position of the internal probe, they fulfill the optimal conditions for carrying out a TaqMan in particular PCR.
  • oligonucleotides described here for the first time can be used in a wide range of different technologies. These include:
  • This method represents a new means of quantifying bacterial populations in ecological research.
  • TaqMan-PCR contains, this PCR assay is suitable for a
  • oligonucleotides described here can be used as probes and primers and are designed in such a way that they can be used in hybridization methods and PCR methods, in particular in TaqMan PCR.
  • the invention also encompasses its complementary sequences and the reverse and reverse complementary sequences and the R A sequences derived therefrom.
  • derivatives of the oligonucleotides described here are also included.
  • “Derivatives and” modifications * are understood to mean those sequences in which at least one nucleotide, but also two or three nucleotides, at one or both ends of the oligonucleotides or inside the oligonucleotides, have been replaced or deleted by other oligonucleotides.
  • the prerequisite here is that specific binding to the 16S rDNA or 16S rRNA of bacteria can take place.
  • oligonucleotides described can also be incorporated into larger DNA and RNA units, for example in vectors and plasmids, and further oligonucleotides can be attached to one or both of the ends of the oligonucleotides. for example with specific restriction enzyme recognition parts.
  • DNA and RNA-directed probes are used for the specific detection of a microorganism are known per se.
  • DNA-directed probe Bach et al. 1999, Appl. And Environ. Microbiol .
  • RNA Bach et al. 1999, J. Microbiol. Methods.
  • the person skilled in the art can use these methods known per se with the present oligonucleotides.
  • the DNA or the RNA of a sample to be examined for the presence of said bacterial genes is brought into contact with at least one of the oligonucleotides which have been described in more detail above.
  • the detection of the hybridization of the oligonucleotides with the RNA or the DNA of the sample is then carried out by means of PCR techniques or by hybridization reactions in order to show the presence of specific DNA and / or RNA sequences. All of the disclosed oligonucleotides can also be used as primers for reverse transcription of mRNA.
  • probes and primers here also called oligonucleotides for short.
  • oligonucleotides for short.
  • a label for example a radioactive label, digoxigenin, peroxidase or alkaline phosphatase-detectable label, or a fluorescent label to detect a specific hybridization.
  • Primers and probes can also be labeled with biotin, for example, and used for sequence-specific isolation of DNA or RNA by means of magnetic capture hybridization.
  • all labels are possible that do not interfere with the detection methods, for example labels that can be detected by enzymatic methods, for example the above-mentioned detection by alkaline phosphatase.
  • probes are e.g. PNAs in which the heterocyclic bases are bound to a protein backbone and not to a sugar-phosphate backbone.
  • the oligonucleotides are bound to a matrix (reverse hybridizations), and hybridization is carried out with, for example, fluorescence-labeled DNA or a PCR amplifier.
  • matrices are microchips and microtiter plates.
  • Figure 1 shows a calibration curve, the creation of which is explained in more detail below under the heading “TaqMan-PCR for the quantification of the -16S rDNA genes from bacteria in liquid culture *.
  • the DNA or RNA of the sample to be examined is either isolated from the sample organisms or the organisms are suitably digested or made permeable so that direct contact of the probes with the DNA and / or the RNA of the sample organisms is made possible without extensive and time-consuming cleaning procedures have to be carried out.
  • the oligonucleotides of the invention can be used for in situ hybridizations and in situ PCR.
  • the hybridization of the oligonucleotides with the DNA and / or the RNA of the sample organisms takes place under stringent conditions, preferably highly stringent conditions. These conditions are detailed below.
  • reaction buffer and wash buffer are composed of the following functional components:
  • Buffer system for adjusting and stabilizing the pH between 7 and 8 e.g. Tris / HCl
  • Components e) and f) influence the binding strengths of nucleic acid duplex molecules. Increasing the monovalent cations in the reaction or washing solution stabilizes the duplex molecules formed, while with an increasing content of e.g. Formamide the duplex bonds are weakened.
  • a suitable oligonucleotide concentration must be used.
  • the hybridization must take place at a suitable temperature (the higher the temperature, the weaker the binding of the hybrids).
  • Stringent hybridization and washing conditions are the reaction conditions (the right choice of the four factors), among which only duplex molecules between oligonucleotides and desired target molecules (perfect hybrids) are created or only the desired target organism is detected.
  • Stringent reaction conditions are understood to mean, for example, a hybridization temperature of approximately 5-10 ° C. below the respective oligonucleotide melting point.
  • the stability of the DNA / DNA or RNA / DNA hybrids must be guaranteed even at low salt concentrations corresponding to 0.1 x SSC / 0.5% SDS. In this way, undesirable cross-reactions with other genes can be prevented.
  • the respective temperature conditions can differ depending on the selected test conditions and depending on the nucleic acid sample to be examined and must then be adapted accordingly.
  • the hybridization product can be detected, for example, by autoradiography in the case of radioactively labeled primer molecules or by fluorimetry when using fluorescence-labeled oligonucleotides.
  • stringent conditions are listed in the examples below.
  • the person skilled in the art can adapt the conditions to the selected examination method in a manner known per se, in order to actually achieve stringent conditions and to enable a specific detection method.
  • Suitable stringency conditions can be determined, for example, using reference hybridizations.
  • the oligonucleotides are used as forward and reverse primers for a PCR reaction.
  • the PCR method has the advantage that very small amounts of DNA can be detected.
  • the temperature conditions and the cycle numbers of the PCR have to be modified.
  • the optimal reaction conditions can be determined by hand tests in a manner known per se.
  • An example of a PCR is as follows:
  • the characteristic, species-specific DNA marker fragments formed in the course of the PCR amplification by extending the primer sequences can be detected, for example, by gel electrophoresis or fluorimetry using fluorescence-labeled oligonucleotides. Of course, other detection methods known to the person skilled in the art can also be used.
  • the probes with the DNA or RNA sample to be examined are used Hybridization brought, whereby stringent hybridization conditions are chosen. Stringent conditions must be determined empirically for the respective application. The conditions described below can serve as guidelines.
  • the DNA or RNA of the sample to be examined can be present either in the PCR reaction or RT-PCR or in the hybridization reaction either in extracted form or in the form of complex mixtures in which the microorganism DNA or RNA to be examined is present forms only a very small fraction of the fraction of the special biological sample.
  • the cells to be examined can thus either be in a purified form or, for example, contaminated with other constituents. Both in situ PCR and in situ RT-PCR can be carried out with the oligonucleotides described here.
  • the oligonucleotides of the present invention are used for the PCR amplification of fragments on cDNA matrices, which are generated after reverse transcription with oligo (dT) or with these oligonucleotides at the 3 V end.
  • the expression can then be evaluated qualitatively, in conjunction with suitable standards and techniques, such as an internal standard and in particular with a quantitative PCR.
  • oligonucleotides can also be used as probes on DOT BLOTS or
  • the oligonucleotides of the invention are produced in a manner which is customary and known per se.
  • the probes can for example inserted into vectors and cut out again after propagation or they can be produced in the form of a template by PCR or synthetically.
  • oligonucleotides described in this application are outstandingly suitable for use in TaqMan PCR.
  • TaqMan * is a registered trademark.
  • the basics of TaqMan-PCR are described below.
  • the specific application of the oligonucleotides according to the invention in the context of a TaqMan PCR is described in the context of an example.
  • PCR polymerase chain reaction
  • a special fluorogenic probe which consists of an oligonucleotide, the 5 ⁇ end of which carries a quencher dye (rhodamine derivative) and is also blocked with a phosphate residue. Modifications to these configurations are of course possible.
  • the fluorescence of the reporter dye is suppressed by a fluorescence energy transfer (FET) due to the spatial proximity to the quencher.
  • FET fluorescence energy transfer
  • the probe first hybridizes with the primers to the template strand. Hits in the extension phase the Taq polymerase now on this probe and begins to displace it. A Y-shaped secondary structure is formed, which activates the 5 ⁇ -3 ⁇ exonuclease activity of the AmpliTaq DNA polymerase and cuts the probe. Free, non-hybridized probe, however, is not hydrolyzed.
  • a TaqMan PCR probe has the following characteristics:
  • the fluorescent reporter dye is covalently linked to the 5 ⁇ end of the probe.
  • FAM is used as a reporter.
  • Table 1 These different reporter dyes, which also allow a multiplex application of the TaqMan PCR system, are each quenched by the quencher dye TAMRA.
  • TAMRA is bound to the 3 ⁇ end of the probe via a linker-modified nucleotide (LAN).
  • LAN linker-modified nucleotide
  • the probe is chemically phosphorylated to avoid extension of the 3 end during the PCR. Further refinements by Use of other reporters and quencher dyes or 'other polymerases are possible and are within the skills and knowledge of the skilled person.
  • the T m of the probe should be at least 5 ° C above that the PCR
  • Primers are, preferably not a 72 ° C extension step, but better 2-
  • the TaqMan TM probe is usually used in concentrations in the range of 50-200 nM / 50 ⁇ l reaction.
  • the optimal probe concentration depends on the fluorescence background and the primer concentration.
  • the reporter fluorescence signal of the without template controls should be at least three times as high as the signal of the buffer blank.
  • the usual primer concentrations in the TaqMan TM -PCR are 0.2 to 0.5 ⁇ M. However, it is recommended to determine the actual optimal concentration by testing the primers in the range of 0.1 to 1.0 ⁇ M.
  • the number of PCR cycles required to obtain a sufficient signal depends on the amount of template molecules used at the start of the PCR. Around 10,000 start copies and 25-30 cycles are sufficient to start optimization.
  • PCR additives such as glycerin and / or formamide
  • annealing temperatures of the primers and the probe should first be determined according to the nearest neighbor method. When optimizing the reaction, the actual annealing temperature must then be e.g. 2 ° C intervals are determined empirically.
  • the duration of the extension depends on the length of the PCR product to be generated:
  • the extension rate of the AmpliTaq-Gold is between 2000 and 4000 bases per minute at temperatures from 70 ° C to 80 ° C.
  • the extension temperature ⁇ 70 ° C in order not to additionally destabilize the hybridization of the probe and thus to achieve higher R n + values.
  • 16S rDNA sequences were obtained from the NCBI database. Alignments were made using the Geno atix DiAlign program (http: // genomatix. Gsf. De / cgi-bin / dialign / dialign.pl). Comparison of the degenerate oligonucleotides designed in this way with known DNA sequences using the Genotamix Matinspektor program
  • DNA from bacteria and fungi to determine the specificity of the oligonucleotides directed against the 16S rDNA was extracted using standard methods (Marmur 1961; Henrion et al. 1994) or obtained from Sigma, Deisenhofen, Germany. DNA from pure cultures for a subsequent TaqMan PCR quantification was extracted using the QIAamp tissue kit (Quiagen, Hilden, Germany). DNA from Pseudomonas fluorescens was extracted according to the instructions from Quiagen. In order to achieve efficient lysis, the method was used using Bacillus spp.
  • the pellets were resuspended in 1 ml lysis buffer and transferred into 2 ml glass tubes, each containing 2 g of glass beads with a diameter of 0.17-0.18 ⁇ (Braun, Melsungen, Germany). During the 30 minute incubation at 37 ° C., the suspensions were homogenized 3 times 90 seconds after 0, 15 and 30 minutes in a Bead Beater (homogenizer) (Braun, Melsungen, Germany) at 2000 rpm. DNA from the soil was extracted and purified using the FastDN ⁇ SPIN kit for soils (Bio 101, Vista, USA) according to the manufacturer's instructions.
  • Plasmid standards were prepared as follows The corresponding 16S rDNA gene fragment was prepared by conventional PCR using the primers shown in Table 2, with sticky ends being added to the primers at the 5 ⁇ ends, namely the restriction recognition sequence for BamHI with the overhang CGC forward primer and restriction recognition sequence for Xbal with overhang -GC- to the reverse primer. Genomic DNA from E. coli was used as a template. The fragment thus obtained was digested and cloned into the high copy vector Triple Helix TM pHelix TM vector 1 (+) (Röche, Mannheim, Germany) and transformed into E.coli JM 105. The plasmid DNA was purified using the plasmid midi kit (Qiagen, Hilden, Germany).
  • the quantitative determination of the plasmid DNA was estimated by comparing the bands with Lambda DNA / EcoRI + HindIII marker 3 (MBI Fermentas, Vilnius, Lithuania) on agarose gel after ethidium bromide staining. 10-fold dilutions were made and used as external standards in TaqMan-PCR.
  • the primers and probes used are given in Table 2.
  • the amplification was carried out using the ABI 7700 sequence detection system (Perkin Elmer, Norwalk, Connecticut, USA) in 50 ⁇ l final volumes with 5 ⁇ l template DNA, 200 nM per primer, 150 nM TaqMan probe, 200 pM deoxynucleotide triphosphate, 5 ml 10 ⁇ reaction buffer , 4.5 mM MgCl 2 and 1.25 U ⁇ mpli Taq Gold DNA polymerase (Perkin Elmer).
  • the PCR program was as follows: a stop at 95 ° C for 10 minutes to denature the DNA and activate the polymerase, 35 cycles at 95 ° C for 15 seconds and 62 ° C for 60 seconds. Results
  • DSMZ 1048 Escherichia coli (genomic DNA, Sigma, Deisenhofen, Germany), Clostridi um perfrigens (genomic DNA, Sigma, Deisenhofen, Germany), Micrococcus l uteus (genomic DNA, Sigma, Deisenhofen, Germany).
  • genomic DNA from calf thymus, Mucor mucedo DSMZ. 809, Aspergill ⁇ s niger DSMZ 1988, Penicilli u funiculosum DSMZ 1960 or Irpex lacteus DSMZ 1183 no products were obtained.
  • Standard plasmid DNA with 2 x 10 7 - 1 x 10 3 copies of the 16S rDNA fragment from E. coli in 10-fold and simple dilutions were used to set up the calibration curve shown in Fig. 1. Two replicates were measured for each sample.
  • the preferred oligonucleotides of the invention are listed in Table 2, along with the preferred reaction conditions under which these oligonucleotides. can be used to quantify bacteria in TaqMan PCR. Temperature program:
  • the reagents and tubes used come from Applied Biosystems, with the exception of the dNTPs used, which were obtained from Röche.
  • the DNA fragment to be amplified from Escherichia coli was inserted into the high copy number pUC19 vector (DNA plasmid, pHelix 1 (+), Röche Mannheim, Germany) inserted and cloned.
  • a PCR was carried out with the primers listed in Table 2, but each was extended at the 5 'end by sticky ends (forward primer: overhang (5 "CGC) and restriction site for SamHI (5 V GGATCC); reverse primer: overhang ( 5 N GC) and restriction site for Xhal (5 "TCTAGA).
  • the product from this PCR and the vector were digested with the two restriction enzymes and combined in a ligation.
  • the vector thus modified was then multiplied and extracted in E. coli exact determination of the concentration of this vector, which contains a copy of the DNA section to be amplified, dilutions were made, of which the number of copies was known and which were used as standards for the TaqMan PCR.
  • the crucial parameter when quantifying DNA copies is the Ct value. This indicates the number of the PCR cycle in which the fluorescence signal rises above the background signal for the first time.
  • Fig. 1 shows the standard curve generated from the standards (black points) and the sample values (red points) entered on them. Due to the high reproducibility of the approach, the individual points for the replicas are fused together and therefore not all are visible.
  • the bacterial concentrations determined via the PCR correspond exactly to those from the DAPI counts.
  • the KBE values are somewhat lower at all sampling times, which is due to the fact that this determination method is fraught with some uncertainties. It could be that either not all organisms grow on agar plates or that bacterial chains, which are particularly common in the exponential growth phase, form only one colony at a time.
  • Oligonucleotides which were used as primers or as probes for the detection and quantification of the bacterial 16S rDNA copies.

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Abstract

La présente invention concerne des oligonucléotides qui peuvent être utilisés notamment dans le cadre d'une amplification en chaîne par polymérase TaqMan (PCR TaqMan), afin de permettre une détermination quantitative de bactéries.
EP01913774A 2000-01-31 2001-01-31 Oligonucleotides permettant l'amplification specifique et la detection specifique de genes d'arnr 16s de bacteries Withdrawn EP1252174A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10004147A DE10004147A1 (de) 2000-01-31 2000-01-31 Oligonukleotide zur spezifischen Amplifikation und zum spezifischen Nachweis von 16S-rRNA-Genen von Bakterien
DE10004147 2000-01-31
PCT/EP2001/001010 WO2001057055A2 (fr) 2000-01-31 2001-01-31 Oligonucleotides permettant l'amplification specifique et la detection specifique de genes d'arnr 16s de bacteries

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EP1252174A2 true EP1252174A2 (fr) 2002-10-30

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EP (1) EP1252174A2 (fr)
AU (1) AU2001239234A1 (fr)
DE (1) DE10004147A1 (fr)
WO (1) WO2001057055A2 (fr)

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Publication number Priority date Publication date Assignee Title
DE10204858B4 (de) * 2002-02-06 2007-08-02 Fröhlich, Jürgen, Dr. Gensonden zum Nachweis von Spezies der Gattung Oenococcus
DE10244456A1 (de) * 2002-09-24 2004-04-15 Hain Lifescience Gmbh Verfahren zum Nachweis und Differenzierung von Bakterien
WO2004046375A2 (fr) * 2002-11-20 2004-06-03 Pharmacia Italia S.P.A. Methode de detection de micro-organismes dans des produits pharmaceutiques
FR2908785B1 (fr) * 2006-11-17 2012-09-28 Air Liquide Methodes de detection de microorganismes par pcr

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US5334501A (en) * 1989-07-11 1994-08-02 Microprobe Corporation Quantification of bacteria using a nucleic acid hybridization assay
US5210015A (en) * 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
US6093538A (en) * 1992-05-06 2000-07-25 Gen-Probe Incorporated Nucleic acid probes to ureaplasma
US5374718A (en) * 1992-08-26 1994-12-20 Gen-Probe Incorporated Nucleic acid probes to chlamydia pneumoniae
US5654144A (en) * 1995-04-24 1997-08-05 The United States Of America As Represented By The Secretary Of The Army Detection of Yersinia using the polymerase chain reaction
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JP2001521394A (ja) * 1997-04-22 2001-11-06 ババリアン・ノルディック・リサーチ・インスティテュート・アクティーゼルスカブ 病原性大腸菌株検出用のＴａｑＭａｎ▲上ＴＭ▼−ＰＣＲ
FR2763957B1 (fr) * 1997-05-29 2001-09-14 Bio Merieux Procede pour isoler un materiel intracellulaire et procede de traitement d'un materiel nucleique
DE19732086C2 (de) * 1997-07-25 2002-11-21 Univ Leipzig Verfahren zur quantitativen Bestimmung von Eubakterien
DE19822108A1 (de) * 1998-05-12 2000-02-03 Schering Ag Verfahren zur Detektion von Mikroorganismen in Produkten, insbesondere in Arzneimitteln und Kosmetika

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AU2001239234A1 (en) 2001-08-14
DE10004147A1 (de) 2001-08-09
WO2001057055A2 (fr) 2001-08-09

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