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  • 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 present invention relates to a method for genotypically classifying bacteria.
• the commonly used method for classifying bacteria, especially in clinical diagnostics, is based on the phenotypic differences of the organisms.
• the classification is based on activity patterns of various metabolic enzymes, as described, for example, in Holt, JG (ed.): Bergey 's Manual of Systematic Bacteriology, Vol. 1-4, Williams & Wilkins, Baltimore, 1984, 1986, 1989.
• the first approaches to genotypic classification were based on 16S rRNA sequence homologies, which were considered to be representative of the entire genome.
• Other universally used genes that have been used for classification are the genes for the elongation factor G (EF-G) and proton-transporting ATPases (Olsen & Woese, FASEB J. 7 (1993) 113-223).
• the genotypic classification based on 16S rRNA sequence homologies provides a natural, phylogenetic, systematic bacteriology (Woese et al., Proc. Natl. Acad. Sei. USA 87 (1990) 4567-4579).
• a gene for the rapid and error-free identification of bacteria at the species level should meet the following criteria:
• the gene is universally present in all bacteria
• Variations in the sequence of the gene are small between individual strains of one species compared to the variations between different species;
• the gene contains species-specific variations
• the gene is conserved so that detection in different species is possible.
• the rpoB gene also meets criteria 1, 2, 4 and 6 in the list above. So far there are no reports that there is more than one copy of the rpoB gene in one strain, so criterion 3 is also met.
• the object of the present invention is to provide a method for genotypic classification that overcomes the above-mentioned disadvantages of the known methods.
• Sequences of partial regions of at least one gene selected from the group gyrA, gyrB, parC and parE are determined and
• the classification is carried out by comparison with the known sequences of the respective genes of bacteria, where
• the codons for the amino acids Gly-81, Ser-83, Ala-84, Asp-87 according to the numbering of the E. coli gyrA gene are not taken into account for the comparison;
• the codons for the amino acids Asp-420 and Lys-441 according to the numbering of the E. coli parE gene are not taken into account for the comparison.
• the new method is based on the analysis of species and subspecies-specific DNA sequence variations in conserved areas of the genes gyrA, gyrB, parC and parE, which code for subunits of bacterial topoisomerase II (gyrase) and topoisomerase IV.
• the observed reactions essentially concern the third "wobble" position of different codons in these regions, which therefore do not change the amino sequence of the expressed protein in most cases.
• the genes also contain the sections (quinolone resistance-determining regions, QRDR), which contain all mutations previously associated with quinolone resistance.
• the corresponding resistance mutations are often point mutations that only affect individual amino acids.
• these are in particular glycine-81, serine-83, alanine-84 and aspartate-87 based on the numbering of the E. coli gyrA gene.
• the amino acids glycine-78, serine-80, alanine-81 and glutamate-84 are essentially involved in the quinolone resistance.
• the corresponding amino acids are aspartate-426 and lysine-447 in the case of the gyrB gene and aspartate-420 and lysine-441 in the case of the parE gene. Since these mutations are not species-specific, they are not taken into account when classifying the bacteria according to the invention. As far as is known, the genes gyrA and gyrB are present in all bacteria examined so far (parC, ParE - not yet - in mycobacteria), are only available as a simple copy and are highly conserved at the protein level in the specified ranges. It is preferred that at least two genes are used for classification at the same time.
• the sequences are preferably first determined via an amplification step for partial regions of the genes.
• the regions relevant to the classification are amplified.
• the determination can be carried out either by sequencing the amplified regions or by hybrid Sization of the amplified areas with species-specific oligonucleotides, followed by, for example, spectroscopic analysis of the hybridization.
• the species-specific oligonucleotide probes preferably have a length of 8 to 14 nucleotides.
• the array technology in which the complementary oligonucleotides are fixed to a surface can preferably be used for the method according to the invention, the analysis being facilitated by labeling the oligonucleotides with different fluorescent dyes.
• the method according to the invention not only permits the genotypic classification of bacteria, but also a further subdivision at sub-species level, whereby epidemiological relationships between different isolates can also be shown.
• epidemiological relationships between different isolates can also be shown.
• the presence of quinolone resistance mutations in the isolated bacteria can be demonstrated at the same time.
• Nucleic acids with Seq ID no. 1 to 13 and fragments of nucleic acids with a length of at least eight nucleotides can be used as probes for the classification of bacteria.
• the invention furthermore relates to a composition which comprises at least two nucleic acids which are specific for sequences of partial regions of genes selected from the group gyrA, gyrB, parC and parE of bacterial species, and an analysis device for classifying bacteria, the analysis device being nucleic acids contains that are specific for sequences of partial regions of genes selected from the group gyrA, gyrB, parC and parE of bacterial species.
• the invention also relates to the use of this analysis device or the composition mentioned above for the analytical or diagnostic classification of bacteria.
• Figures 1 to 4 show the comparison of the sequences gyrA, gyrB, parC and parE of the different bacterial species with the E. coli K12 sequence. Differences in the sequences compared to the E.coli K12 sequence are printed in bold. Underlines indicate amino acid changes ( Figures 2 to 4).
• the starting point for the examination is a representative single colony that was isolated from the examination material (infection focus) of a patient. This colony is resuspended in sterile water (50 to 100 ⁇ l). By boiling (15 min) template DNA is obtained for a subsequent amplification reaction.
• oligonucleotides are added to this template DNA as primer pairs, as are deoxynucleotide triphosphates, suitable buffers (e.g. for Taq DNA polymerase in the event that a PCR is carried out) and an enzyme (e.g. Taq DNA polymerase).
• suitable buffers e.g. for Taq DNA polymerase in the event that a PCR is carried out
• an enzyme e.g. Taq DNA polymerase
• enterobacteria the following combination of primers for the gyrA or parC genes has proven itself:
• gyrA5- 1 5 '-GAATCCGGGATACAGTAGAGGGATAG-3 '
• gyrA3-l 5 '-CCTTAAACCAACCGTACTGCAGGCCT-3 '
• parC-S 5 '-GTATGCGATGTCTGAACTGGGCCTG-3 '
• parC-U 5 ' -ACCGGGATTCGGTGTAACGCATTGC-3 '
• parE5 ' 5' -GAG CTG TTC CTT GTG GAA GG-3 ' or 5 ' -GAG CTG TTT CTT GTG GAG GG-3 '
• parE3 ' 5 ' -GGT GTT AAG GAT CTT ACC-3 ' or 5 ' -GGT ATT AAG GAC CTT ACC-3 ' gyrB5 ' : 5 ' CTG CCG GGC AAA CTG GC-3 ' or 5 ' CTG CCG GGC AAA CTA GC-3 '
• gyrB3 ' 5 ' -AC GTT GAG GAT TTT ACC-3 ' or 5 ' -AC GTT AAG AAT TTT ACC-3 '
• the DNA fragments obtained by amplification (30 cycles with the following temperature profile 30 s 95 ° C, 30 s 50 ° C; 45 s 72 ° C) are purified by separating the primer, enzyme and template DNA.
• the purified fragment is used for a DNA sequence analysis of the QRDR regions (e.g. by means of hybridization with an array of octamer oligonucleotides, SBH).
• the aim is to identify specific sequence variations, such as those identified in FIGS. 1 to 4 as being characteristic of individual species or subspecies.
• the investigated isolate is then assigned to a species.

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• 2000
  • 2000-04-10 EP EP00926858A patent/EP1169482A1/de not_active Withdrawn
  • 2000-04-10 WO PCT/EP2000/003187 patent/WO2000061796A1/de not_active Application Discontinuation
  • 2000-04-10 AU AU45463/00A patent/AU4546300A/en not_active Abandoned
• 2002
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