WO2004029299A2 - Procede d'identification et de differenciation de bacteries - Google Patents

Procede d'identification et de differenciation de bacteries Download PDF

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WO2004029299A2
WO2004029299A2 PCT/EP2003/010584 EP0310584W WO2004029299A2 WO 2004029299 A2 WO2004029299 A2 WO 2004029299A2 EP 0310584 W EP0310584 W EP 0310584W WO 2004029299 A2 WO2004029299 A2 WO 2004029299A2
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sequences
complementary
nucleic acid
fragment
sequence
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PCT/EP2003/010584
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German (de)
English (en)
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WO2004029299A3 (fr
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Michael Weizenegger
Michaela Bollen
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Hain Lifescience Gmbh
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Priority to AU2003270248A priority Critical patent/AU2003270248A1/en
Priority to EP03750608A priority patent/EP1543151A2/fr
Publication of WO2004029299A2 publication Critical patent/WO2004029299A2/fr
Publication of WO2004029299A3 publication Critical patent/WO2004029299A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to the field of diagnostics of microorganisms, in particular the detection of bacteria which occur in clinically relevant samples.
  • the nucleic acids described here can preferably be used to identify bacteria in cultures, in particular in blood cultures.
  • the invention relates in particular to hybridization methods and amplification methods, as well as coupled amplification / hybridization methods with sequence-specific probes or primers.
  • the cultivation of the bacteria in liquid or solid media is a sensitive standard method for the detection of bacteria in the human primary sample.
  • the differentiation is usually carried out by microscopy of cultural or primary preparations, according to the morphology on surfaces of cultivated colonies, by growth tests under various conditions and by catabolic properties, e.g. the metabolism of sugar, the formation of acids, etc. Due to the necessary installation of secondary and / or tertiary cultures, several days can pass before the findings are made.
  • the object of the present invention was therefore to provide highly specific and highly sensitive ner methods for the detection of clinically important bacteria, the development of primers for carrying out an amplification method for bacterial genome fragments and / or their transcripts and the development of a detection system carrying many probes. These tests preferably run in the multiplex method.
  • a ner method for the detection of clinically relevant bacteria in which a nucleic acid to be detected, which is a fragment from the genome of a clinically relevant bacterium or is complementary to this, hybridizes to a sequence- and / or species-specific nucleic acid probe under stringent conditions and then the nucleic acid to be detected or the hybridization of the nucleic acid to be detected to the sequence-specific nucleic acid probe is detected, characterized in that the sequence-specific nucleic acid probe is selected from the sequences with SEQ ID No .: 1-176 or is complementary to these sequences, or a fragment represents or is complementary to this fragment, one contains these sequences or the complementary sequence or is a variant thereof.
  • nucleic acid and oligonucleotide refers to primers, samples, probes and oligomer fragments which are detected.
  • nucleic acid and oligonucleotide is also generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose) and polyribonucleotides (containing D-ribose) or to any other type of polynucleotide that is an N-glycoside of a purine base or a pyrimidine base, respectively a modified purine base or a modified pyrimidine base.
  • PNAs are also included according to the invention, i. H. Polyamides with purine / pyrimidine bases.
  • nucleic acid and oligonucleotide are not considered to be different within the meaning of the present invention; in particular, the use of the terms is not intended to mean any differentiation in terms of length. These terms include both double and single stranded DNA and double and single stranded RNA.
  • sequence-specific nucleic acid probe is selected from the sequences with SEQ ID No .: 19-54 and 174-176 or is complementary to these sequences, or represents a fragment thereof or is complementary to this fragment or one of these sequences or contains the complementary sequence.
  • probes which differ slightly from the probes according to the invention, but which can nevertheless be used according to the invention. It is also conceivable to use probes that have the sequences SEQ ID no. 1-176 or 19-54 and 174-176 at the 5 'and / or 3' end of the truncations by one, two or three nucleotides.
  • a nucleic acid probe which contains one of the SEQ ID No.:l-176 or a sequence complementary thereto preferably comprises nucleic acid probes which at the 5 'and or 3' ends 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10 contains additional nucleotides, these 5 'and / or 3' flanking sequences preferably having homology to the respective natural 5 'and / or 3 'flanking sequences of SEQ ID No .: 1-176, more preferably 19-54 and 174-176 of at least about 50%, 55%, 60%, 65%, 70%, 75%,
  • flanking sequences are preferably chosen so that they do not significantly impair the hybridization of the nucleic acid probe. However, the specificity is preferably increased by the additional flanking nucleotides.
  • a variant of SEQ ID No .: 1-176, preferably SEQ ID No .: 19-54 and 174-176, which can be used according to the invention is a nucleic acid probe in which 1, 2, 3, 4, 5, 6, 7, 8, 8, 10 nucleotides compared to the one identified by SEQ ID No. 1-176 specific sequence have been changed. Preferably no more than 1 to 4, 1 to 3, 1 to 2, and most preferably only one nucleotide are changed. Such a nucleotide change is preferred if the specificity of the probe is not significantly changed thereby and / or the melting point of the probe is not significantly changed thereby. The specificity of a probe modified in this way can easily be checked by the person skilled in the art by routine experiments.
  • a variant in the sense of the invention is present when the probe is to be detected with respect to the particular one.
  • Nucleic acid at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more than 100% of the specificity of the unchanged nucleic acid probe according to SEQ ID No .: 1-176.
  • composition which contains the nucleic acid to be detected or a part thereof, with one, preferably at least two, three, four, five, six, seven, eight, new, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or hybridized more different probes.
  • the nucleic acid to be detected it is possible to determine the nucleic acid to be detected and thus, for example, the bacterial species by hybridization with a single specific probe.
  • more than one probe preferably two, for the composition which contains the nucleic acid to be detected or a part thereof or three to hybridize. This increases the informative value of the process.
  • An exact profile is then obtained and the nucleic acid to be detected and thus, for example, the bacterial species can be determined with a high degree of certainty.
  • a preferred method for the differentiation of clinically relevant bacteria is therefore a multiplex method in which the bacterial genome sections necessary for the differentiation are enriched and hybridized in a common amplification and hybridization reaction.
  • the amplification of various gene fragments is necessary for this application.
  • the probe and primer for the detection of the bacterial species are complementary to 23 S ribosomal RNA genes and / or to the elongation factor TU, the probes and primers for differentiating the antibiotic resistance have the corresponding genomically coded resistance genes as the target sequence. Since the polymorphisms necessary for differentiation on the genome encompass many 1000 bases, a splitting into smaller fragments is also important for an effective amplification.
  • the variety of primers that can all interact with one another requires careful design.
  • a multiplex amplification method uses at least two different ones
  • Nuclear acid fragments amplified simultaneously Preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more different nucleic acid sequences are amplified simultaneously.
  • twice as many primers are used, ie at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 and more primers.
  • a multiplexed hybridization method preferably a reverse hybridization method, is preferably connected to the multiplex amplification method immediately or after further intermediate steps such as, for example, cleaning steps.
  • a hybridization matrix according to the invention has a corresponding one Number of different immobilized probes.
  • Another important aspect is a multiplexing method with a self-amplifying chip.
  • probes with different primary structures have to hybridize under the same conditions, whereby the specificity should be sufficient for the detection of single-base pockets. For this reason, salts such as betaine or tetramethylammomumchloid are preferred as modulators of the hydrogen bonds in the stringency washing.
  • Preferred polymerases are thermostable "hot start polymerases for the self-amplifying chip:
  • Preferred buffers are PCR buffers which contain modulating chemicals such as Preferred amplification primers are selected from SEQ ID No. 1-176, or complementary thereto, or a fragment thereof or contain one of these sequences, particularly preferred are SEQ ID No .: 55-170.
  • salts such as betaine or Tetramethylammomumchloid preferred, but also stabilizing proteins like BSA.
  • hybridization refers to the formation of duplex structures by two single-stranded nucleic acids due to complementary base pairing. Hybridization can take place between complementary strands of nucleic acid or between strands of nucleic acid which have small mismatch ranges.
  • the stability of the nucleic acid duplex is measured by the melting temperature T m .
  • the melting temperature T m is the temperature (at defined ionic strength and pH) at which 50% of the base pairs are dissociated.
  • Stringent hybridization conditions are known to the person skilled in the art (for example Sambrook et al., 1085, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). In general, stringent conditions are selected so that the melting temperature is 5 ° C lower than the T m for the specific sequence at a defined ionic strength and pH. If the hybridization is performed under less stringent conditions, then sequence mismatches are tolerated. The The extent of sequence mismatches can be controlled by changing the hybridization conditions.
  • Stringent in the sense of the present invention means that the detection method allows a clear distinction between a positive reaction and a negative reaction in the reaction field of the strip. This can be achieved through the following measures:
  • Structure of the probe by the length of the structure of the probe complementary to the target sequence; 15 to 20 mers are preferred.
  • Running buffer The stringency is influenced by the salinity.
  • the ionic strength is preferably between about 100-500 mM, more preferably between about 150-350 mM, more preferably between 200-300 mM and most preferably about 250 mM.
  • the stringency can be changed by adding one of the mildly denaturing substances (DMSO, formamide, urea) mentioned above to the running buffer.
  • DMSO mildly denaturing substances
  • the stringency is also influenced by the pH value of the running buffer.
  • all of the above-mentioned measures are measures which have an influence on the formation of hydrogen bonds.
  • solid phase or hybridization matrix encompasses any material that is able to form covalent or non-covalent bonds to nucleic acid probes such as, for example, glass, SiO 2 , plastics such as nylon and filter materials such as nitrocellulose.
  • the materials can also be chemically functionalized to form a bond to nucleic acid probes
  • the shape in particular the surface shape, is in no way limited and includes smooth and structured surfaces
  • Hybridization is preferably carried out on a nylon or nitrocellulose membrane, as described, for example, in (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1989). The principles mentioned therein can be transferred to further embodiments by the person skilled in the art.
  • the length of the target nucleic acid also plays an important role in the sensitivity of the hybridization. Nucleic acid strands with a length of 30-500 base pairs are preferred.
  • the double-stranded target nucleic acid is denatured before hybridization. This is usually done using basic chemicals or heating, whereby the hydrogen bonds responsible for the double-strand structure are melted.
  • a preferred basic chemical is NaOH in a concentration of about 0.1 to 0.5 M. A concentration of about 0.25 M NaOH is particularly preferred.
  • Single-strand structures can also be achieved by heating an aqueous nucleic acid solution to at least 95 ° C. and then rapidly cooling to 4 ° C.
  • Single-strand certificates, e.g. B. as products of the NASBA reaction should also be denatured before hybridization in order to resolve intramolecular structures. Due to the sensitivity of the RNA to high pH values, this can preferably be caused by mildly denaturing chemicals such as DMSO or formamide.
  • Aqueous buffers with a salt content of between 0.1 and 0.5 M and a pH of 7.5-8.0 are generally used as hybridization buffers.
  • Detergents are used to ensure good wetting of the probe-carrying phase.
  • Sodium lauryl sulfate (SDS) is preferred in a concentration of 0.1-7%. In the particularly preferred high concentration of 7%, SDS improves the signal / background ratio by suppressing non-specific bindings, for example of an enzyme complex coupled to the nucleic acid to be detected.
  • the desired stringency of the hybridization is determined by the composition of the hybridization and stringency washing buffer. After hybridization, incubation with a stringency wash buffer is preferred. This destabilizes the double strand through a lower ionic strength. This means that hybrids that are not 100% complementary are separated again.
  • Chemicals e.g. tetramethylammonium chloride
  • the extent of the hybridization is determined after the hybridization. This is usually done by determining the amount of label that is bound to a solid phase.
  • detection reactions and detection methods are known per se to the person skilled in the art.
  • a preferred embodiment of the method according to the invention is characterized in that the nucleic acid to be detected is an amplification product, the amplification being carried out with sequence-specific amplification primers.
  • the nucleic acid to be detected is an amplification product, at least one amplification primer being selected from the sequences with the SEQ ID No .: 1-173, in particular SEQ ID No. 1-18 and 171-173, or is complementary to these sequences, or represents a fragment thereof or is complementary to this fragment or contains one of these sequences or the complementary sequence.
  • At least one amplification primer is selected from the sequences with SEQ ID No .: 1-18 and 55-170 and 171-173 or is complementary to these sequences, or is a fragment thereof or is complementary to this fragment or contains one of these sequences or the complementary sequence.
  • the amplification primers should be selected so that the amplification product has good steric ratios in combination with the immobilized probe. Pallindrome structures that lead to intramolecular folds can be avoided by selecting suitable primers.
  • the spatial arrangement of the hapten e.g. biotin
  • the hapten should be easily accessible to the antibody-enzyme complex.
  • An embodiment of the method is characterized in that the nucleic acid probes are immobilized. In this case it is advantageous if the nucleic acid to be detected is marked. In another form of the method, the nucleic acid probes are labeled. In this case it is advantageous if the nucleic acid to be detected is immobilized.
  • the invention further relates to a method for the detection of clinically associated bacteria
  • a nucleic acid to be detected which is a fragment from the genome of a clinically relevant bacterium or is complementary to it, is amplified, the amplification being carried out with primers, at least one of which has a sequence which essentially represents a partial sequence of the nucleic acid to be detected .
  • the sequence of this primer is selected from the sequences with SEQ ID No .: 1-176 or is complementary to these sequences, or is a fragment thereof or is complementary to is this fragment or contains one of these sequences or the complementary sequence.
  • This embodiment of the invention is briefly called the amplification method below.
  • the term amplification method includes all preferred embodiments.
  • primers can also be designed which differ slightly from the primers according to the invention, but can nevertheless be used according to the invention. It is also conceivable to use primers which have the sequences SEQ ID no. 1-18 or 55-170 and 170-173 at the 5 'and / or 3' end of the truncations by one, two or three nucleotides.
  • flanking sequences are preferably chosen so that they do not significantly impair the hybridization of the nucleic acid primer. However, the specificity is preferably increased by the additional flanking nucleotides.
  • a variant of SEQ ID No .: 1-176, preferably SEQ ID No .: 19-54 and 174-176, which can be used according to the invention is a nucleic acid probe in which 1, 2, 3, 4, 5, 6, 7, 8, 8, 10 nucleotides compared to the one identified by SEQ ID No. 1-176 specific sequence have been changed. Preferably no more than 1 to 4, 1 to 3, 1 to 2, and most preferably only one nucleotide are changed. Such a nucleotide change is preferred if the specificity of the probe is not significantly changed thereby and / or the melting point of the probe is not significantly changed thereby. The specificity of a probe modified in this way can easily be checked by the person skilled in the art by means of routine experiments.
  • a variant within the meaning of the invention is present when the probe has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 with regard to the particular nucleic acid to be detected %, 100% or more than 100% of the specificity of the unchanged nucleic acid probe according to SEQ ID No .: 1-176.
  • the last-mentioned method is most preferred if at least two primers have a sequence which essentially represents a partial sequence of the nucleic acid to be detected, the sequences of these primers being selected from the sequences with SEQ ID No .: 1-176 or complementary to these Sequences are or represents a fragment thereof or is complementary to this fragment or contains one of these sequences or the complementary sequence.
  • the primer (s) have a sequence which essentially represents a partial sequence of the nucleic acid to be detected, the Sequences of these primers are selected from the sequences with SEQ ID No .: 1-18 and 171-173 and SEQ ID No .: 55-170 or are complementary to these sequences, or represent a fragment thereof or are complementary to this fragment or contains one of these sequences or the complementary sequence.
  • primers are marked.
  • PCR polymerase chain reaction
  • TMA transcriptase mediated amplification
  • RT-PCR reverse transcriptase polymerase chain reaction
  • SDA single strand displacement amplification
  • the amplificate is specifically cut, for example by digestion with a restriction enzyme, and the resulting ethidium bromide-stained fragments are analyzed on an agarose gel.
  • Hybridization systems are also widespread. Hybridization usually takes place in such a way that either the composition which contains the amplification product or a part thereof or the probe is immobilized on a solid phase and brought into contact with the other hybridization partner in each case.
  • materials are conceivable as solid phases, for example nylon, nitrocellulose, polystyrene, silicate materials etc. It is also conceivable that a microtiter plate is used as the solid phase.
  • the target sequence can also hybridize with a capture probe beforehand in solution and then the capture probe is bound to a solid phase.
  • at least one probe or at least one primer is labeled during the amplification of the nucleic acid to be detected.
  • labels are conceivable, such as fluorescent dyes, biotin or digoxigenin.
  • Known fluorescent labels are fluorescein, cyanine dyes, etc.
  • the labels are usually covalently linked to the oligonucleotides. While fluorescent labeling can be detected directly, biotin and digoxigenin labeling can be detected after incubation with suitable binding molecules.
  • a biotin-labeled oligonucleotide can be detected by contacting it with a solution that contains streptavidin coupled to an enzyme, the enzyme, for example peroxidase or alkaline phosphatase, converting a substrate that produces a dye or leads to chemical luminescence ,
  • the enzyme for example peroxidase or alkaline phosphatase, converting a substrate that produces a dye or leads to chemical luminescence
  • the nucleic acid probes are immobilized on the solid phase, and then this solid phase is brought into contact with the composition which contains the labeled nucleic acids to be detected or a part thereof.
  • the composition which contains the labeled nucleic acids to be detected or a part thereof Preferably at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or more different probes are immobilized on the solid phase, more preferably at least five different probes, more preferably at least ten different probes. Different probes can be immobilized in different zones.
  • the solid phase is therefore preferably a microarray of immobilized probes on a solid phase.
  • the solid phases which are suitable for DNA chips preferably consist of silicate materials such as glass etc.
  • the marking of the primers is preferably a fluorescent marking.
  • the DNA chip After incubation with the amplification product or the sample, the DNA chip can contain the nucleic acid to be detected or a part thereof can be quickly analyzed by a scanning device. Such devices are known to the person skilled in the art. McGlennen (2001) Miniaturization technologies for molecular diagnostics gives an overview of the chip technology. Clin Chem 47 (3), 393-402.
  • the nucleic acid to be detected is marked.
  • labels such as fluorescent dyes, biotin or digoxigenin.
  • Known fluorescent labels include fluorescein, FITC, cyanine dyes, rhodamines, rhodamine 6 ooR-phycoerythrin, Texas Red etc.
  • radioactive labeling such as 125 I, 35 S, 32 P, 35 P.
  • Particle labeling is also possible like with latex. Such particles are usually dry, in the micron range and uniform.
  • the labels can be covalently or non-covalently and directly or indirectly connected to the oligonucleotides and are usually linked covalently.
  • a direct link exists if a covalent or non-covalent chemical bond between.
  • a fluorescent label can be detected directly, for example, biotin and digoxigenin labels can be detected after incubation with suitable binding molecules or conjugate partners.
  • Other binding partners than, for example, biotm / streptavidin are antigen / antibody systems, hapten / anti-hapten systems, biotin / avidin, folic acid / folate-binding proteins, complementary nucleic acids, proteins A, G and immunoglobulin etc. (MN Bobrov, et al J. Immunol. Methods, 125, 279, (1989).
  • a biotin-labeled oligonucleotide can be detected by contacting it with a solution that contains streptavidin coupled to an enzyme, the enzyme, for example peroxidase or alkaline phosphatase, converting a substrate that produces a dye or leads to chemical luminescence .
  • the enzyme for this purpose are hydrolases, lyases, oxido reductases, transferases, isomerases and ligases.
  • Other examples are peroxidases, glucose oxidases, phosphatases, esterases, and glycosidases. Such methods are known per se to the person skilled in the art (Wetmur JG.
  • Another preferred conjugate comprises an enzyme which is coupled to an antibody (Williams, J. Immunol. Methods, 79, 261 (1984).
  • nucleic acid to be detected with a gold steptavidin conjugate, in which case a biotin-labeled Oligonucleotide can be detected, but conceivable are also binding partners that form covalent bonds with one another, such as sulfhydryl-reactive groups such as maleimides and haloacetyl derivatives and amine-reactive groups such as isothiocyanates, succinimidyl esters and sulfonyl halides.
  • sulfhydryl-reactive groups such as maleimides and haloacetyl derivatives
  • amine-reactive groups such as isothiocyanates, succinimidyl esters and sulfonyl halides.
  • nucleic acids to be detected are labeled, the probes are usually not labeled.
  • the nucleic acids to be detected are thus essentially labeled using the methods described in the prior art (see also US Pat. No. 6,037,127).
  • the labeling can be introduced into the nucleic acid to be detected by chemical or enzymatic methods, or by direct incorporation of labeled bases into the nucleic acid to be detected.
  • sequences to be detected which have labeled markers are produced by labeled bases or labeled primers during the PCR.
  • Labeled primers can be made by chemical synthesis e.g. using the phosphoramidite method by substituting bases of the primer with labeled phosphoramidite bases during the primer synthesis.
  • primes can be prepared with modified bases to which labels are chemically bound after the primer synthesis.
  • ribosomal RNA species can hybridize specifically with a DNA probe and can be detected as an RNA / DNA hybrid with an RNA / DNA-specific antibody.
  • T4 polynucleotide kinase or a terminal transferase enzyme Another possibility is the introduction of labels using the T4 polynucleotide kinase or a terminal transferase enzyme.
  • the introduction of radioactive or fluorescent labels (Sambrook et. Al, Molecular Cloning, Cold Spring Harbor Laboratory Press, Vol. 2, 9.34-9.37 (1989); Cardullo et. al. PNAS, 85, 8790; Morrison, Anal. Biochem, 174, 101 (1988).
  • Markers can be introduced into one or both ends of the nucleic acid sequence of the nucleic acid to be detected. Markers can also be introduced within the nucleic acid sequence of the nucleic acid to be detected. Several markings can be introduced into a nucleic acid to be detected.
  • At least one of the probes has a marking.
  • the composition containing the amplification product or a part thereof is then immobilized on a solid phase and brought into contact with a composition which contains at least one probe.
  • several solid phases can be provided on which the amplification product or the sample containing the nucleic acid to be detected is immobilized.
  • the present invention furthermore relates to a device for the detection of clinically relevant bacteria comprising a solid phase on which one or more sequence- and / or species-specific nucleic acid probes are immobilized, characterized in that the sequence-specific nucleic acid probe is selected from the sequences with the SEQ ID No .: 1-176 or is complementary to these sequences, or represents a fragment thereof or is complementary to this fragment or contains one of these sequences or the complementary sequence.
  • sequence-specific nucleic acid probe is selected from the sequences with SEQ ID No .: 19-54 and 174-176 or is complementary to these sequences, or represents a fragment thereof or is complementary to this fragment or one of these Contains sequences or the complementary sequence. If several oligonucleotides are immobilized, they are spatially separated from one another on the solid phase.
  • the solid phase is preferably designed as a DNA chip.
  • the solid phase is a self-amplifying DNA chip that has at least two or more oligonucleotides immobilized at its 5 'ends in each application unit (“spot”). These act as amplification primers.
  • the specificity of these immobilized primers is special due to their structure biologically defined in the region of the 3 'region.
  • This DNA chip is therefore not a hybridization chip, but a nucleic acid amplification chip.
  • This is analogous to multiplex amplification, which contains more than two primers in one approach and thus delivers more than one amplification product
  • the amplification reaction taking place in the reaction vessel is severely limited in its multiplexing ability. 30 to 60 amplification products in one reaction currently represent a technical maximum.
  • the multiplexing ability of the nucleic acid amplification chip is only limited by the chip area n some 10,000 spots per cm 2 are applied here. This results in the possibility of using primers in a reaction complementary to any positions of a complete genome as the target molecule.
  • the present invention thus relates to a DNA chip which has immobilized amplification primers.
  • Preferred amplification primers are selected from SEQ ID No. 1-176 or complementary to these sequences, or a fragment thereof or complementary to this fragment or contains one of these sequences or the complementary sequence, SEQ ID No .: 55-170 are particularly preferred for this object of the invention.
  • the solid phase of the device according to the invention can be a chromatographic material. Since the analyte is mainly hydrophilic in nature, hydrophilic properties of the chromatographic material of the test strip are important for carrying out the method according to the invention.
  • the chromatographic material may include inorganic powders such as silicate materials, magnesium sulfate and aluminum, may further comprise synthetic or modified naturally occurring polymers such as nitrocellulose, cellulose acetate, cellulose, polyvinyl chloride or acetate, polyacrylamide, nylon, cross-linked dextran, agarose, polyacrylate, etc. may further include coated materials such as ceramic materials and glass. Most preferred is the use of nitrocellulose as the chromatographic material.
  • the introduction of positively charged ion groups in eg nitrocellulose or nylon membranes can improve the hydrophilic properties of the chromatographic material.
  • the chromatographic material can be mounted in a housing or the like.
  • This housing is usually water-insoluble, rigid and can consist of a variety of organic and inorganic materials. It is important that the housing does not interfere with the capillary properties of the chromatographic material, that the housing does not bind test components non-specifically, and that the housing does not interfere with the detection system.
  • a coated glass carrier is preferably used as the solid phase for the self-amplifying chip.
  • Such glass supports are known to the person skilled in the art.
  • the device according to the invention is preferred if the sequence- and / or species-specific nucleic acid probes are bound to the solid phase of the device via a linker.
  • the linker acts as a spacer between the probe and the membrane. In the present case, these are mostly polymers which extend the part of the probe which is complementary to the target sequence at the 5 'or 3' end, but are not themselves coding. These can be base sequences of a non-coding nucleic acid structure or other polymer units such as polyether, polyester etc.
  • the linker must be such that it does not affect the hybridization properties of the probe or only weakly. This can be avoided by the fact that there are no self-complementary structures. The chemical prerequisites for the irreversible coupling of the probe to the carrier material must also be met.
  • Probe oligonucleotides can, for example, be bound to the membrane surface via proteins.
  • the proteins loaded with the probe can then be bound to the porous membrane by standard methods. Standard methods are, for example, coupling via homobifunctional coupling reagents or heterobifunctional coupling reagents.
  • the reactive groups are the same. Typically these are amines and / or thiols. Thiols can be synthetically coupled directly to oligonucleotides and react under oxidative conditions with cysteine residues to form disulfide bridges.
  • amines as homobifunctional coupling reagents can be directly synthetically coupled to oligonucleotides and bound to the surface or the protein via imido esters or succinimide esters. With heterobi-functional coupling reagents, the reactive groups are different and allow the coupling of different functional groups. The formation of amino-thiol couplings is preferred.
  • Thiolated oligonucleotides can be coupled with a heterobifunctional coupling reagent which contains both a succimmide ester maleimide or iodoacetimide.
  • Another important coupling agent are the carbodiimides, which couple carbonyl residues to amines. The most important representative here is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC).
  • EDAC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • amino modified oligonucleotides can be coupled to membranes with carbonyl residues. With this chemistry, the coupling reagent is not incorporated into the compound.
  • the hybridization of the probes can also be carried out “in solution” as a homogeneous method.
  • the probes can be modified such that they are labeled with dyes (eg rhodamines, fluoresces and their derivatives) in combination with light-suppressing (“quenchers”) molecules are distinguishable in their hybridized and free states.
  • dyes eg rhodamines, fluoresces and their derivatives
  • quenchers light-suppressing
  • a nucleic acid is selected from the sequences with SEQ ID No .: 1-176 or is complementary to these sequences, or is a fragment thereof, or is complementary to this fragment or contains one of these sequences or the complementary sequence.
  • the present invention furthermore relates to a composition or a kit for the detection of clinically relevant bacteria containing one or more of the nucleic acids according to the invention.
  • the subject of the present invention is a kit for the amplification of a nucleic acid to be detected, which is one or more fragments from the genome of a clinically relevant bacterium or complementary thereto, containing one or more of the nucleic acids according to the invention.
  • the kit contains all necessary for the amplification of the target sequence
  • Components such as primers, buffer systems and enzymes and the immobilized probes for the detection and specification of the amplificate with the necessary buffer systems.
  • kits for the amplification of a nucleic acid to be detected which is a fragment from the genome of a clinically relevant bacterium or complementary to it, containing one or more nucleic acids selected from the sequences with the SEQ ID No .: 1-18, 171 - 173 and 55-170 or complementary to these sequences, or a fragment thereof, or complementary to this fragment or containing one of these sequences or the complementary sequence.
  • the invention also relates to the use of the device according to the invention for the detection of clinically relevant bacteria.
  • the invention further relates to the use of the nucleic acid according to the invention or the kit according to the invention for the detection of clinically relevant bacteria.
  • the nucleic acid to be detected can be in any composition that is suspected of containing bacteria, in particular clinically relevant bacteria. It can be primary material such as secretions, sulcus fluid, smears and blood etc. Or Cultures of microorganisms that have already been grown in liquid or solid media.
  • HIN Haemophilus influencae
  • PMI Proteus mirabilis
  • Seq ID No. 20 Serratia marcescens (SMA) Seq ID No. 21, Escherichia coli (ECO) Seq ID No. 22, Pseudomonas pudica (PPU) Seq ID No. 24, Pseudomonas fluorescens (PFL) Seq ID No. 25, Acinoetobacter baumanii (ABA) Seq ID No. 26, Klebsieila oxytoca (KOX) Seq ID No. 27, Klebsieila pneumoniae (KPN) Seq ID No. 28, Enterobacter aerogenes
  • EAE Seq ID No. 29
  • Pseudomonas aerogenosa Seq ID No. 30
  • PVU Proteus vulgaris
  • BCE Burgholderia cepacia
  • Seq ID No. 34 Staphylococcus capitis (SCA) Seq ID No. 35, Enterococcus faecalis (EFA) Seq ID No. 36, Enterococcus durans (EDU) Seq ID No. 37, Staphylococcal Group F (SGF) Seq ID No. 38, Enterococcus faecium (EFC) Seq ID No. 39, Enterococcus durans (EDU) Seq ID No. 40, Streptococcus pyogenes (SPY) Seq ID No. 41, Streptococcus sciuni (SCI) Seq ID No. 42
  • Streptococcus oralis Seq ID No. 43, Staphylococcus saprophyticus (PVU) Seq ID No. 44, Staphylococcus cohaii (SCO) Seq ID No. 45, Staphylococcus lugdunensis / haemolyticus / hominis (SLHH) Seq ID No. 46, Staphylococcus warneri (SWA) Seq ID No. 47, Staphylococcus epidermidis (SEP) Seq ID No. 48, Staphylococcus lyticans (SLY) Seq ID No. 49, Staphylococcus grinding (SOR) Seq ID No. 50;
  • Positive band strip 1 hybridizes with amplified DNA from Staphylococcus aureus: biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 33
  • Positive band strip 2 hybridizes with amplified DNA from Staphylococcus epidermidis: biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 48
  • Positive band strips 3 hybridized with amplified Enterococcus faecium DNA biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 39
  • Positive band strips 5 hybridized with amplified Enterococcus faecalis DNA biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 36
  • Positive band strip 6 hybridizes with amplified DNA from Streptococcus pyogenes: biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 41
  • Positive band strip 7 hybridizes with amplified DNA from Enterococcus gallinarum: biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 34
  • Positive band strips 8 hybridized with amplified Enterococcus casseliflavus DNA biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 56
  • Positive band strips 9 hybridize with amplified Enterococcus durans DNA: biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 40
  • Positive band strips 10 hybridized with amplified DNA from Staphylococcus saprophyticus biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 44
  • Positive band strip 11 hybridizes with amplified DNA from Staphylococcus cohnii: biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 45
  • Positive band strip 12 hybridizes with amplified DNA from Staphylococcus warneri: biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 47
  • Positive band strips 13 hybridize with amplified DNA from Staphylococcus lyticans: biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 49
  • Positive band strips 14 hybridized with amplified DNA from Staphylococcus loops: biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 50
  • Positive band strips 15 hybridize with amplified DNA from Enterococcus casseliflavus: biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 56, SEQ ID No .: 54 positive band strips 16 hybridize with amplified DNA from Enterococcus faecalis: biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 36, SEQ ID No .: 51 Positive band strips 17 hybridized with amplified Enterococcus gallinarum DNA: biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 34, SEQ ID No .: 53 positive bands strip 18 hybridized with amplified Enterococcus faecium DNA: biotinylated DNA, SEQ ID No .: 55, SEQ ID No .: 39, SEQ ID No .: 52
  • FIG. 4 Analysis example of a self-amplifying chip.
  • the glass carrier was coated with the following prime pairs:
  • the corresponding bacteria can be identified and differentiated either from primary material (e.g. smears, blood, etc.) or from bacterial liquid or solid media.
  • Bacterial nucleic acid was obtained either from solid nutrient media, liquid media or from primary material after appropriate pretreatment.
  • bacterial material was removed from solid media with a sterile inoculation loop and suspended in 300 ⁇ l lOmM Tris / HCl pH 7.5. 1 ml was removed from liquid cultures, centrifuged for 5 min at 13,000 ⁇ m in a table centrifuge, the supernatant was discarded and resuspended in 300 ⁇ l lOmM Tris / HCl pH 7.5. The bacterial suspension was incubated for 15 minutes at 95 ° C.
  • thermomixer Eppendorf, Hamburg, Germany
  • sonicated for 15 minutes in an ultrasonic bath Bandelin, Berlin,
  • centrifuged for 10 minutes at 13,000 ⁇ m in a table centrifuge Eppendorf, Hamburg, Germany
  • 5 ⁇ l of the supernatant were used in the amplification reaction.
  • the PCR approach contained 1 x Taq buffer (Qiagen, Hilden, Germany), each 1 ⁇ M primer, 200 ⁇ M dNTP (Röche, Mannheim, Germany) and 1U Hotstar Taq polymerase (Qiagen, Hilden, Germany).
  • the PCR amplification was carried out on a PE 9600 thermocycler (ABI, Rothstadt, Germany) at 15 minutes at 95 ° C., 30 cycles at 20 seconds at 95 ° C. and 30 seconds at 60 ° C. Detection of the amplificates by probe hybridization:
  • probes were biotinylated at the 5 'end in order to be able to detect target sequence / probe hybrids via reporter enzymes coupled to steptavidin. Oligonucleotides with the sequences Table 1 SEQ ID NO 19-22, 24-50 are used as probes.
  • Absorbent paper (blotting paper GB002, Schleicher & Schüll, Dassel, Germany) and a nylon membrane (Biodyne A, Pall, Portsmouth, England) were cut to the size of the blot apparatus (Minifold Schleicher & Schüll, Dassel, Germany) and cut with 10 x SSC soaked. 250 ⁇ l of denaturing solution (50 mM NaOH; 1.5 M NaCl) were placed in the openings of the assembled apparatus and 20 ⁇ l of amplificate were pipetted in. After applying a vacuum, it was waited until all the liquid had been sucked through completely. It was then rinsed with 10 ⁇ SSC buffer. After it had dried completely, the membrane was fixed in a UV crosslinker (UV-Stratalinker 2400, Stratagene, La Jolla, USA) at 1200 joules / cm 2 and washed with distilled water and dried.
  • UV crosslinker UV crosslinker
  • the hybrids were autoradiographically detected by a streptavidin-alkaline phosphatase conjugate by adding NBT / BCIP or by spraying on chemiluminescent substrate (Lumi-Phos 530, Cellmark Diagnostics, Abindon, England). Streptavidin-alkaline phosphatase conjugate was added and incubated at 37 ° C for 30 min. The membrane was then washed twice with substrate buffer for 15 min. The membrane was then removed, Lumi-Phos reagent was sprayed on, followed by 2 h exposure to an X-ray film. Alternatively, substrate buffer with NBT / BCIP was added and the color development awaited. Solutions used:
  • 10 x SSC solution (standard saline citrate): 1.5M NaCl, 0.15M trisodium citrate;
  • TMCL tetramethylammonium chloride
  • NBT 75 mg / ml nitroblue tetrazolium salt in 70% dimethylformamide
  • the autoradiograms were evaluated densitometrically.
  • the amplificate dot of the species from which the probe sequence was derived was used as the 100% value.
  • a sample that was added to water instead of nucleic acid solution and a sample with 100 ng of isolated human DNA were always carried as dots on the membrane.
  • the results of Example 1 are shown in Figures 1 and 2.
  • The% values of the densitometric evaluation are given.
  • the value of the probe homologous to the species was set to 100%.
  • Blot apparatus cut and impregnated with 10 x SSC 250 ⁇ l of a 1 ⁇ M probe solution, as described in FIG. 3, were placed in the openings of the assembled apparatus in 10 ⁇ SSC in slot-like openings in the blot apparatus. After applying a vacuum, it was waited until all the liquid had been sucked through completely. It was then rinsed with 10 ⁇ SSC buffer. After it had dried completely, the membrane was fixed in a UV crosslinker (UV-Stratalinker 2400, Stratagene, La Jolla, USA) at 1200 joules / cm 2 and washed with distilled water, dried and cut into strips about 4 mm wide.
  • UV crosslinker UV-Stratalinker 2400, Stratagene, La Jolla, USA
  • the hybrids were colorimetrically detected by a streptavidin-alkaline phosphatase conjugate with the addition of NBT / BCIP. Streptavidin-alkaline phosphatase conjugate was added and incubated at 37 ° C for 30 min. The membrane was then washed twice with substrate buffer for 15 min. The membrane was incubated in substrate buffer with NBT / BCIP 10min and color development stopped by washing with H 2 Obide st (See Fig. 3).
  • Example 3 Self-amplifying chip
  • the species-specific amino-modified ohgonucleotide pairs (Interactiva, Ulm, Germany) are diluted in Na-phosphate buffer 20 ⁇ M and pipetted into a plate with 384 wells.
  • the ohgonucleotide pairs are applied with a volume of lnl in round fields ("spots") with a diameter of 200 ⁇ m each side by side on the glass support (75 x 25 mm).
  • spots lnl in round fields
  • the pair of ohgonucleotides are always immobilized in duplicate side by side (duplicate spots).
  • the amino-modified oligonucleotide pairs (Interactiva, Ulm, Germany) are immobilized on the surface-modified glass supports in all cases by “contact-p ⁇ ' ⁇ t g” with the help of the OMNI-GRJD (GeneMachines, San Carlos, USA) spotters.
  • the species-specific oligonucleotides are removed from the wells of the microtitre plate by a robot arm provided with a series of precision tips and placed on precisely defined positions on the glass slides.
  • the "Motorola Activated slides" chip blanks (Motorola, Schaumburg, Illinois USA) have reactive groups on their surface that can covalently bind amino-modified oligonucleotides to the surface (no further information from the manufacturer).
  • the spotted glass slides are incubated overnight in a humid chamber with saturated sodium chloride solution at room temperature.
  • the glass supports 15 Incubated for minutes at 50 ° C in blocking reagent and washed 2 x 5 minutes at room temperature in deionized water. This is followed by a 15 minute incubation at 50 ° C in the Motorola washing buffer and again the glass slides are washed in deionized water for 2 minutes at room temperature.
  • the glass slides are dried by centrifugation at 10000 m for 5 minutes.
  • the glass slides are removed from the thermal cycler and the cover slip is carefully removed.
  • the glass slides are washed for 15 minutes at 68 ° C. in wash buffer 1, 5 minutes at room temperature in wash buffer 2 and 5 minutes at room temperature in wash buffer 3. They are then dried using compressed air.
  • the glass slides are then scanned with the aid of a confocal laser scanner (Scan Array 4000, DSS Imagetech, New Delhi, India) and the result is the visual representation of the fluorescence signal on the glass slide.
  • a confocal laser scanner Scan Array 4000, DSS Imagetech, New Delhi, India.
  • the more intense the fluorescence signal on the glass substrate the whiter the respective spot appears and, conversely, the weaker the signal, the blacker the respective spot appears.
  • the colors red-green-blue appear on the intensity scale between the white and black spots.
  • the fluorescence signals were integrated into numerical values using the ImaGene software (ImaGene Standard edition; Biodiscovery, Marina del Ray, USA) (see Figure 4).
  • the primers SEQ ID No. 55-170 are designed as species-specific amplification primers for the self-amplifying chip.

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Abstract

L'invention concerne le domaine du diagnostic de micro-organismes, notamment l'identification de bactéries. L'invention concerne notamment des procédés d'hybridation et d'amplification, ainsi que des procédés d'amplification/d'hybridation couplés, mis en oeuvre à l'aide de sondes spécifiques de séquences ou d'amorces.
PCT/EP2003/010584 2002-09-24 2003-09-23 Procede d'identification et de differenciation de bacteries WO2004029299A2 (fr)

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Cited By (6)

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WO2005054516A3 (fr) * 2003-11-26 2006-03-09 Advandx Inc Sondes d'acides nucleiques peptidiques pour l'analyse de certaines especes staphylococcus
WO2009090310A1 (fr) * 2008-01-17 2009-07-23 Mobidiag Oy Procédé de détection et d'identification de staphylocoques
EP2106450A1 (fr) * 2007-01-08 2009-10-07 Medigenes Co., Ltd. Puce d'adn permettant la détection d'escherichia coli
US7888075B2 (en) 2007-07-31 2011-02-15 Quest Diagnostics Investments Incorporated Detection of methicillin-resistant and methicillin-sensitive Staphylococcus aureus in biological samples
US8097409B2 (en) 2005-02-07 2012-01-17 Gen-Probe Incorporated Kits for detecting group B Streptococci
US9109260B1 (en) 1999-03-02 2015-08-18 King's College London Identification of bacteria by amplification and probing

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US9109260B1 (en) 1999-03-02 2015-08-18 King's College London Identification of bacteria by amplification and probing
WO2005054516A3 (fr) * 2003-11-26 2006-03-09 Advandx Inc Sondes d'acides nucleiques peptidiques pour l'analyse de certaines especes staphylococcus
US8097409B2 (en) 2005-02-07 2012-01-17 Gen-Probe Incorporated Kits for detecting group B Streptococci
EP2106450A1 (fr) * 2007-01-08 2009-10-07 Medigenes Co., Ltd. Puce d'adn permettant la détection d'escherichia coli
EP2106450A4 (fr) * 2007-01-08 2010-03-10 Medigenes Co Ltd Puce d'adn permettant la détection d'escherichia coli
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US7888075B2 (en) 2007-07-31 2011-02-15 Quest Diagnostics Investments Incorporated Detection of methicillin-resistant and methicillin-sensitive Staphylococcus aureus in biological samples
WO2009090310A1 (fr) * 2008-01-17 2009-07-23 Mobidiag Oy Procédé de détection et d'identification de staphylocoques

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