EP1038035A1 - VERFAHREN ZUR IDENTIFIZIERUNG VON $i(ESCHERICHIA COLI) STAMM DSM 6601 - Google Patents

VERFAHREN ZUR IDENTIFIZIERUNG VON $i(ESCHERICHIA COLI) STAMM DSM 6601

Info

Publication number
EP1038035A1
EP1038035A1 EP98966241A EP98966241A EP1038035A1 EP 1038035 A1 EP1038035 A1 EP 1038035A1 EP 98966241 A EP98966241 A EP 98966241A EP 98966241 A EP98966241 A EP 98966241A EP 1038035 A1 EP1038035 A1 EP 1038035A1
Authority
EP
European Patent Office
Prior art keywords
strain dsm
escherichia coli
dna
dna sequences
identifying
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Withdrawn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
EP98966241A
Other languages
German (de)
English (en)
French (fr)
Inventor
Jörg Hacker
Ulrich Sonnenborn
Gabriele Blum-Oehler
Jürgen Schulze
Jürgen Malinka
Hans Proppert
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.)
Pharma Zentrale GmbH
Original Assignee
Pharma Zentrale 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 Pharma Zentrale GmbH filed Critical Pharma Zentrale GmbH
Publication of EP1038035A1 publication Critical patent/EP1038035A1/de
Withdrawn legal-status Critical Current

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 invention relates to a method for identifying Escherichia coli (E. coli) strain DSM 6601.
  • Escherichia coli is a gram-negative bacterium that is found in the human and animal intestinal flora, but also extraintestinal. E. coli is today the most important host organism among the microbial cloning systems of genetic engineering for the expression of heterologous proteins as well as for cloning and DNA amplification.
  • E. coli occurs in numerous variants, which differ in terms of capsule antigens (K antigens), surface antigens (O antigens) and flagella antigens (H antigens) and can therefore be divided into numerous serological types.
  • K antigens capsule antigens
  • O antigens surface antigens
  • H antigens flagella antigens
  • the classification according to the serotypes says nothing about the different virulence of the pathogens.
  • Representatives of one and the same serotype can have different pathogenicity potential in both the human and animal body, which in extreme cases can range from avirulent to highly pathogenic. It is known that the E. coli strain DSM 6601 is rated as non-human or animal pathogenic.
  • a method for identifying E. coli strain DSM 6601 is now proposed, which is characterized in that certain primer pairs from the plasmids or the fimA and foc ⁇ sequences of the bacterial DNA are used in a PCR reaction.
  • the PCR polymerase chain reaction
  • the detection method according to the invention is based on that of RK Saiki et al. methods described in Science 239: 487-491 (1988).
  • primers that is, oligonucleotides, which generally have a length of about 15 to 30 nucleotides and whose sequences are complementary to the start and end sequences of the sister strands of the DNA to be amplified, are required.
  • the double-stranded DNA of the sequence to be amplified is denatured by heating so that it separates into single strands.
  • the complementary strand is formed later in the course of the single-stranded region of the nucleic acid referred to as a template or template.
  • the mixture with the primers is cooled, the primer nucleotides hybridizing at the ends of the single-stranded DNA and thereby preventing the original single-stranded DNA from reuniting. This is followed by increasing the Temperature a mixture of the four DNA-typical nucleotide 5'-triphosphates and a temperature-stable DNA polymerase.
  • the Taq polymerase from the extremely thermophilic organism Thermus aquaticus has proven to be particularly suitable and can withstand brief heating to over 95 ° C. At 72 ° C, the polymerase supplements the DNA single strand between the two ends filled with primers to form the double strand.
  • the three process steps namely heat denaturation, primer annealing and polymerization, can be repeated until the batch is exhausted. Since the amount of DNA is doubled in each individual step, a multiplication factor of about 10 is theoretically achieved after about 20 cycles.
  • the primer pairs used are those from the fimA sequence with the designation Muta 1 and 2 (FIG. 1) and those from the focA sequence with the designation Muta 3 and 4 (FIG. 2) from the strain DSM 6601. These DNA sequences are partially in agreement with genes from other enterobacteria, but on the other hand there are bases at some positions that have not been observed in other enterobacteria.
  • the other primer pairs Muta 5 and 6 (Fig. 3), Muta 7 and 8 and Muta 9 and 10 (Fig. 4) were derived from the DNA sequences of the plasmids pMUT 1 (Fig. 5) and pMUT 2 (Fig. 6) of the strain DSM 6601 selected. These primer pairs also have a nucleotide sequence that has not previously been found in enterobacteria. The sequences of the primers Muta 1 to Muta 10 are shown in detail in the attached Figures 1 to 4.
  • a colony of E. coli strain DSM 6601 is inoculated from an agar plate and suspended in 100 ⁇ l bidistilled water. This suspension is heated to 95 ° C. for 10 minutes and then cooled on ice. 1 ⁇ ⁇ of the bacterial suspension is used as template DNA for the PCR.
  • PCR reaction mixture is then pipetted into a PCR reaction tube:
  • the steps b. to d. are repeated at least 20 times.
  • the end products can then be used, for example, for the identification of Escherichia coli strain DSM 6601 or can also be sequenced in a manner known per se and used for checking correspondingly produced DNA sequences from E. coli strains to be examined.

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)
EP98966241A 1997-11-19 1998-11-18 VERFAHREN ZUR IDENTIFIZIERUNG VON $i(ESCHERICHIA COLI) STAMM DSM 6601 Withdrawn EP1038035A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19751243 1997-11-19
DE19751243 1997-11-19
PCT/EP1998/007398 WO1999025870A1 (de) 1997-11-19 1998-11-18 Verfahren zur identifizierung von escherichia coli stamm dsm 6601

Publications (1)

Publication Number Publication Date
EP1038035A1 true EP1038035A1 (de) 2000-09-27

Family

ID=7849197

Family Applications (1)

Application Number Title Priority Date Filing Date
EP98966241A Withdrawn EP1038035A1 (de) 1997-11-19 1998-11-18 VERFAHREN ZUR IDENTIFIZIERUNG VON $i(ESCHERICHIA COLI) STAMM DSM 6601

Country Status (9)

Country Link
US (1) US6489107B1 (enExample)
EP (1) EP1038035A1 (enExample)
JP (1) JP2004516801A (enExample)
CZ (1) CZ289739B6 (enExample)
EE (1) EE200000231A (enExample)
HU (1) HUP0004409A3 (enExample)
NO (1) NO20002550L (enExample)
PL (1) PL190847B1 (enExample)
WO (1) WO1999025870A1 (enExample)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109576198A (zh) * 2018-11-09 2019-04-05 南京工业大学 一株敲除fimA基因的重组大肠杆菌及其构建方法与应用

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19915772C2 (de) * 1998-11-18 2001-08-30 Pharma Zentrale Gmbh Verfahren zur Identifizierung von Escherichia coli Stamm DSM 6601
US20050064447A1 (en) * 2001-04-18 2005-03-24 Sheng-He Huang Probiotic therapy of neonatal meningitis and method of using E. coli virulence determinatns
DE10155928A1 (de) * 2001-11-15 2003-06-12 Degussa recA-negativer und rhaB-negativer Mikroorganismus
EP3506917A4 (en) 2016-08-31 2020-05-06 President and Fellows of Harvard College MANIPULATED BACTERIA PRODUCING THERAPEUTIC PROTEINS AND METHOD FOR USE THEREOF
WO2022060848A1 (en) * 2020-09-15 2022-03-24 Northeastern University Plasmid vectors for in vivo selection-free use with the probiotic e. coli nissle

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19713543B4 (de) * 1997-04-02 2007-01-11 Pharma-Zentrale Gmbh Bakterielle Plasmide
DE19751242C2 (de) 1997-11-19 2001-02-08 Pharma Zentrale Gmbh DNA-Sequenzen aus Fimbriengenen von Escherichia coli Stamm DSM 6601

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9925870A1 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109576198A (zh) * 2018-11-09 2019-04-05 南京工业大学 一株敲除fimA基因的重组大肠杆菌及其构建方法与应用
CN109576198B (zh) * 2018-11-09 2022-06-07 南京工业大学 一株敲除fimA基因的重组大肠杆菌及其构建方法与应用

Also Published As

Publication number Publication date
NO20002550L (no) 2000-07-18
CZ20001873A3 (cs) 2000-10-11
JP2004516801A (ja) 2004-06-10
PL340330A1 (en) 2001-01-29
NO20002550D0 (no) 2000-05-18
US6489107B1 (en) 2002-12-03
EE200000231A (et) 2001-06-15
CZ289739B6 (cs) 2002-03-13
HUP0004409A3 (en) 2001-09-28
HUP0004409A2 (hu) 2001-04-28
PL190847B1 (pl) 2006-02-28
WO1999025870A1 (de) 1999-05-27

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