WO2000061796A1 - Procede de classification genotypique - Google Patents

Procede de classification genotypique Download PDF

Info

Publication number
WO2000061796A1
WO2000061796A1 PCT/EP2000/003187 EP0003187W WO0061796A1 WO 2000061796 A1 WO2000061796 A1 WO 2000061796A1 EP 0003187 W EP0003187 W EP 0003187W WO 0061796 A1 WO0061796 A1 WO 0061796A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
comparison
sequences
bacteria
parc
Prior art date
Application number
PCT/EP2000/003187
Other languages
German (de)
English (en)
Inventor
Peter Heisig
Yolanda Fuchs-Gomez
Original Assignee
Merlin Gesellschaft Für Mikrobiologische Diagnostika Mbh
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
Priority claimed from DE19916227A external-priority patent/DE19916227A1/de
Priority claimed from EP99116340A external-priority patent/EP1077265A1/fr
Application filed by Merlin Gesellschaft Für Mikrobiologische Diagnostika Mbh filed Critical Merlin Gesellschaft Für Mikrobiologische Diagnostika Mbh
Priority to EP00926858A priority Critical patent/EP1169482A1/fr
Priority to AU45463/00A priority patent/AU4546300A/en
Publication of WO2000061796A1 publication Critical patent/WO2000061796A1/fr
Priority to HK02103680.7A priority patent/HK1042117A1/zh

Links

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

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.

Landscapes

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

Abstract

L'invention concerne un procédé de classification génotypique de bactéries, caractérisé en ce que des séquences de zones partielles d'au moins un gène, sélectionnées dans le groupe gyrA, gyrB, parC et parE, sont déterminées, et la classification est réalisée par comparaison avec des séquences connues des gènes respectifs de bactéries. Dans le cas du gène gyrA, les codons pour les acides aminés Gly-81, Ser-83, Ala-84, Asp-87 selon la numérotation du gène E.coli-gyrA, ne sont pas pris en considération pour la comparaison ; dans le cas du gène parC, les codons pour les acides aminés Gly-78, Ser-80, Ala-81, Glu-84 selon la numérotation du gène E.coli-parC ne sont pas pris en considération pour la comparaison ; dans le cas du gène gyrB, les codons pour les acides aminés Asp-426 et Lys-447 selon la numérotation du gène E.coli-gyrB ne sont pas pris en considération pour la comparaison, et dans le cas du gène parE, les codons pour les acides aminés Asp-420 et Lys-441 selon la numérotation du gène E.coli-parE ne sont pas pris en considération pour la comparaison.
PCT/EP2000/003187 1999-04-10 2000-04-10 Procede de classification genotypique WO2000061796A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP00926858A EP1169482A1 (fr) 1999-04-10 2000-04-10 Procede de classification genotypique
AU45463/00A AU4546300A (en) 1999-04-10 2000-04-10 Genotypic classification method
HK02103680.7A HK1042117A1 (zh) 1999-04-10 2002-05-15 基因型分類方法

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE19916227.1 1999-04-10
DE19916227A DE19916227A1 (de) 1999-04-10 1999-04-10 Verfahren zur genotypischen Klassifizierung
EP99116340.3 1999-08-19
EP99116340A EP1077265A1 (fr) 1999-08-19 1999-08-19 Procede de classification de bacteries par l'etablissement du genotype

Publications (1)

Publication Number Publication Date
WO2000061796A1 true WO2000061796A1 (fr) 2000-10-19

Family

ID=26052846

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2000/003187 WO2000061796A1 (fr) 1999-04-10 2000-04-10 Procede de classification genotypique

Country Status (4)

Country Link
EP (1) EP1169482A1 (fr)
AU (1) AU4546300A (fr)
HK (1) HK1042117A1 (fr)
WO (1) WO2000061796A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1288312A2 (fr) * 2001-09-04 2003-03-05 Osaka Prefectural Institute of Republic Health Procédé de détermination de la résistance au quinolone de bacilles de tubercule
WO2004046379A1 (fr) * 2002-11-19 2004-06-03 Mobidiag Oy Sondes d'acide nucleique et amorces a large spectre provenant de regions de genes de la topoisomerase, et procedes utilisant celles-ci
WO2009090311A2 (fr) * 2008-01-17 2009-07-23 Mobidiag Oy Sondes d'acides nucléiques et amorces à large portée

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001584A1 (fr) * 1992-07-06 1994-01-20 President And Fellows Of Harvard College Procedes et necessaires de diagnostic pour determiner la toxicite utilisant des promoteurs de stress bacteriens fusionnes a des genes rapporteurs
EP0688873A2 (fr) * 1994-06-23 1995-12-27 Bayer Ag Test DNA rapide pour la détection de staphylococcus aureus résistant à la chinolone dans du matériel clinique
US5645994A (en) * 1990-07-05 1997-07-08 University Of Utah Research Foundation Method and compositions for identification of species in a sample using type II topoisomerase sequences
WO1999050458A2 (fr) * 1998-04-01 1999-10-07 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Sondes d'oligonucleotides de detection d'enterobacteriaceae et d'enterobacteriaceae resistants aux quinolones

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5645994A (en) * 1990-07-05 1997-07-08 University Of Utah Research Foundation Method and compositions for identification of species in a sample using type II topoisomerase sequences
WO1994001584A1 (fr) * 1992-07-06 1994-01-20 President And Fellows Of Harvard College Procedes et necessaires de diagnostic pour determiner la toxicite utilisant des promoteurs de stress bacteriens fusionnes a des genes rapporteurs
EP0688873A2 (fr) * 1994-06-23 1995-12-27 Bayer Ag Test DNA rapide pour la détection de staphylococcus aureus résistant à la chinolone dans du matériel clinique
WO1999050458A2 (fr) * 1998-04-01 1999-10-07 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Sondes d'oligonucleotides de detection d'enterobacteriaceae et d'enterobacteriaceae resistants aux quinolones

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BACHOUL R ET AL: "Analysis of the mutations involved in fluoroquinolone resistance of in vivo and in vitro mutants of Escherichia coli", MICROBIAL DRUG RESISTANCE, vol. 4, no. 4, 1998, pages 271 - 6, XP002118442 *
BEBEAR ET AL.: "Cloning and nucleotide sequences of the topoisomerase IV parC and parE genes of Mycoplasma hominis", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 42, no. 8, August 1998 (1998-08-01), XP002133970 *
GUILLEMINE ET AL.: "Correlation between quinolone susceptibility patterns and sequences in the A and B subunits of DNA gyrase in mycobacteria", ANTIMICROB. AGENTS AND CHEMOTHERAPY, vol. 42, no. 8, 1998, pages 2084 - 2088, XP002133967 *
HUANG: "Bacterial diversity based on type II DNA topoisomerase genes", ANNU.REV.GENET., vol. 30, 1996, pages 79 - 107, XP000891519 *
KUMAGAI ET AL.: "Quinolone-resistant mutants of E.coli DNA topoisomerase IV parC gene", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 40, no. 3, March 1996 (1996-03-01), pages 710 - 714, XP002133969 *
MARGERRISON ET AL: "Nucleotide Sequence of the Staphylococcus aureus gyrB-gyrA Locus Encoding the DNA Gyrase A and B Proteins", JOURNAL OF BACTERIOLOGY,US,WASHINGTON, DC, vol. 174, no. 5, 1 March 1992 (1992-03-01), pages 1596 - 1603, XP002087511, ISSN: 0021-9193 *
NISHINO Y ET AL: "Mutations in the gyrA and parC genes associated with fluoroquinolone resistance in clinical isolates of citrobacter freundii", FEMS MICROBIOLOGY LETTERS, vol. 154, no. 2, 1997, pages 409 - 14, XP002118439 *
WEIGEL L ET AL: "gyrA mutations associated with fluoroquinolone resistance in eight species of enterobacteriaceae", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 42, no. 10, October 1998 (1998-10-01), pages 2661 - 67, XP002118443 *
YAMADA ET AL.: "Cloning and nucleotide sequence analysis of gyrB of Bacillus cereus, B.thuringiensis, B.mycoides, and B.anthracis and their application to the detection of B.cereus in rice", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 65, no. 4, April 1999 (1999-04-01), pages 1483 - 1490, XP002133968 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1288312A2 (fr) * 2001-09-04 2003-03-05 Osaka Prefectural Institute of Republic Health Procédé de détermination de la résistance au quinolone de bacilles de tubercule
EP1288312A3 (fr) * 2001-09-04 2003-04-23 Osaka Prefectural Institute of Republic Health Procédé de détermination de la résistance au quinolone de bacilles de tubercule
WO2004046379A1 (fr) * 2002-11-19 2004-06-03 Mobidiag Oy Sondes d'acide nucleique et amorces a large spectre provenant de regions de genes de la topoisomerase, et procedes utilisant celles-ci
WO2009090311A2 (fr) * 2008-01-17 2009-07-23 Mobidiag Oy Sondes d'acides nucléiques et amorces à large portée
WO2009090311A3 (fr) * 2008-01-17 2009-11-12 Mobidiag Oy Sondes d'acides nucléiques et amorces à large portée

Also Published As

Publication number Publication date
AU4546300A (en) 2000-11-14
HK1042117A1 (zh) 2002-08-02
EP1169482A1 (fr) 2002-01-09

Similar Documents

Publication Publication Date Title
DE60038015T2 (de) Verfahren, basierend auf einer polynukleotidmatrix, zur identifizierung von mikroorganismen
AT503862B1 (de) Pathogen-identifizierung anhand eines 16s oder 18s-rrna mikroarray
Overmann et al. Phylogeny and molecular fingerprinting of green sulfur bacteria
DE69532675T2 (de) Verfahren zum nachweis des antibiotikaresistenzspektrums von mykobakterium spezies
CN101023170B (zh) 结核菌检测用的引物和探针以及使用它们检测人型结核菌的方法
EP2985350B1 (fr) Procédé d'analyse de microbiome
WO1999058713A2 (fr) Procede de detection de micro-organismes presents dans des produits
WO2001007648A1 (fr) Procede permettant de detecter des organismes de façon specifique a l'espece
DE69722028T2 (de) Genetische marker und verfahren zur erkennung von listeria monocytogenes und listeriaspezien
DE69735561T2 (de) Sonden zur Erkennung und Identifizierung von Helicobacter Pylori
EP1169482A1 (fr) Procede de classification genotypique
DE102005002672A1 (de) Verfahren zur schnellen Differenzierung von Mikroorganismen auf Subspezies-Ebene mittels MALDI-TOF MS
EP0574852A2 (fr) Détection spécifique du Neisseria gonorrhoeae
DE69009744T3 (de) Sonden, ausrüstungen und verfahren zum nachweis und zur differenzierung von mycobakterien
DE10215238C1 (de) Nachweis von Mykobakterien in klinischem Material
EP2373813B1 (fr) Procédé destiné à détecter des souches de staphylocoque doré résistant à la méthicilline (sarm)
EP1077265A1 (fr) Procede de classification de bacteries par l'etablissement du genotype
EP0744469B1 (fr) PCR inter LINE
DE19916227A1 (de) Verfahren zur genotypischen Klassifizierung
DE69631413T2 (de) Nachweis einer bakterie der pectinatus gattung
DE19616750A1 (de) Verfahren zum Nachweis von Mikroorganismen in Gemischen
EP1390541B1 (fr) Procede de detection de bacteries gram positif
EP1606420B1 (fr) Procede et kit pour detecter m. tuberculosis de maniere specifique
Hollaway et al. Use of fatty acid profiles and repetitive element polymerase chain reaction (PCR) to assess the genetic diversity of Pseudomonas syringae pv. pisi and Pseudomonas syringae pv. syringae isolated from field peas in Australia
EP1335991B1 (fr) Procede pour detecter des protozoaires du genre naegleria

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2000926858

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2000926858

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Ref document number: 2000926858

Country of ref document: EP