EP1038035A1 - METHOD FOR IDENTIFYING $i(ESCHERICHIA COLI) STRAIN DSM 6601 - Google Patents

METHOD FOR IDENTIFYING $i(ESCHERICHIA COLI) STRAIN DSM 6601

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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
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EP
European Patent Office
Prior art keywords
strain dsm
escherichia coli
dna
dna sequences
identifying
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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.)
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Application number
EP98966241A
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German (de)
French (fr)
Inventor
Jörg Hacker
Ulrich Sonnenborn
Gabriele Blum-Oehler
Jürgen Schulze
Jürgen Malinka
Hans Proppert
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Pharma Zentrale GmbH
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Pharma Zentrale GmbH
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Application filed by Pharma Zentrale GmbH filed Critical Pharma Zentrale GmbH
Publication of EP1038035A1 publication Critical patent/EP1038035A1/en
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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

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.

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  • 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

The invention relates to DNA sequences with nucleotide sequences as represented in figures 1-4, in addition to a method for identifying escherichia coli strain DSM 6601, whereby the primary pairs from the DNA sequences represented in figures 1-4 are placed in a PCR reaction.

Description

Verfahren zur Identifizierung von Escherichia coli Stamm DSM 6601Procedure for the identification of Escherichia coli strain DSM 6601
Die Erfindung betrifft ein Verfahren zur Identifizierung von Escherichia coli (E. coli) Stamm DSM 6601 .The invention relates to a method for identifying Escherichia coli (E. coli) strain DSM 6601.
Escherichia coli ist ein gramnegatives Bakterium, das in der menschlichen und tierischen Darmflora, aber auch extraintestinal vorkommt. E. coli ist heute unter den mikrobiellen Klonierungssystemen der Gentechnik der wichtigste Wirtsorganismus zur Expression heterologer Proteine sowie zur Klonierung und DNA-Amplifikation.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 tritt in zahlreichen Varianten auf, die sich hinsichtlich der Kapselantigene (K-Antigene), Oberflächenantigene (O-Antigene) und Flagellenantigene (H-Antigene) unterscheiden und daher in zahlreiche serologische Typen unterteilt werden können. Die Einordnung nach den Serotypen besagt allerdings nichts über die unterschiedliche Virulenz der Erreger. Vertreter ein- und desselben Serotyps können sowohl im menschlichen als auch im tierischen Körper unterschiedliches Pathogenitätspotential besitzen, das im Extremfall von avirulent bis hochgradig pathogen reichen kann. Bekannt ist, daß der E.-coli-Stamm DSM 6601 als nicht human- oder tierpathogen bewertet wird.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. However, 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.
Es besteht daher noch ein Bedarf an Verifizierungsmethoden für nichtpa- thogene E.-coli-Stämme. Die Serotypisierung als alleinige Methode zur Beurteilung, ob ein E.-coli-Stamm pathogen oder nichtpathogen ist, reicht nicht aus. Es wurde bereits darauf hingewiesen, daß unter dem gleichen Serotyp sowohl pathogene als auch nichtpathogene Varianten auftreten. Für diagnostische und therapeutische Zwecke in der Medizin, aber auch für den Einsatz zu gentechnologischen Zwecken ist es daher wünschenswert, einzelne Stämme zweifelsfrei identifizieren zu können.There is therefore still a need for verification methods for non-pathogenic E. coli strains. Serotyping as the sole method of assessing whether an E. coli strain is pathogenic or not is not sufficient. It has already been pointed out that both pathogenic and non-pathogenic variants occur under the same serotype. For diagnostic and therapeutic purposes in medicine, but also for the Use for genetic engineering purposes, it is therefore desirable to be able to identify individual strains without any doubt.
Erfindungsgemäß wird nunmehr ein Verfahren zur Identifizierung von E. coli Stamm DSM 6601 vorgeschlagen, das dadurch gekennzeichnet ist, daß in einer PCR-Reaktion bestimmte Primerpaare aus den Plasmiden bzw. den fimA- und focΛ-Sequenzen der Bakterien-DNA eingesetzt werden.According to the invention, 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 focΛ sequences of the bacterial DNA are used in a PCR reaction.
Die PCR (Polymerase chain reaction) ist ein Verfahren, bei dem es gelingt, einige wenige Moleküle einer beliebigen genomischen DNA-Sequenz in kürzester Zeit in vitro um Faktoren von 106 bis 10° zu vermehren. Das erfindungsgemäße Nachweisverfahren ist angelehnt an das von R.K. Saiki et al. in Science 239: 487-491 (1988) beschriebene Verfahren.The PCR (polymerase chain reaction) is a process in which it is possible to multiply a few molecules of any genomic DNA sequence in vitro in a very short time by factors of 10 6 to 10 °. The detection method according to the invention is based on that of RK Saiki et al. methods described in Science 239: 487-491 (1988).
Zur Durchführung der PCR werden Primer, das sind Oligonucleotide benötigt, die in der Regel eine Länge von etwa 15 bis 30 Nucleotiden aufweisen und deren Sequenzen zu den Anfangs- bzw. Endsequenzen der Schwesterstränge der zu amplifizierenden DNA komplementär sind.In order to carry out the PCR, 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.
Zunächst wird die Doppelstrang-DNA der zu amplifizierenden Sequenz durch Erwärmen denaturiert, so daß sie sich in Einzelstränge auftrennt. Über den als Template oder Matrize bezeichneten einzelsträngigen Bereich der Nucleinsäure wird der komplementäre Strang im späteren Verlauf gebildet. Nach der Denaturierung durch Erwärmen kühlt man das Gemisch mit den Primem ab, wobei die Primernucleotide an den Enden der Einzelstrang-DNA hybridisieren und dadurch eine Wiedervereinigung der ursprünglichen DNA- Einzelstränge verhindern. Daran anschließend setzt man nach Erhöhung der Temperatur ein Gemisch der vier DNA-typischen Nucleotid-5'-triphosphate und eine temperaturstabile DNA-Polymerase zu. Als besonders geeignet hat sich die Taq-Polymerase aus dem extrem thermophilen Organismus Thermus aquaticus erwiesen, die auch eine kurzzeitige Erhitzung auf über 95° C übersteht. Bei 72° C wird durch die Polymerase der DNA-Einzelstrang zwischen den beiden mit Primern besetzten Enden zum Doppelstrang ergänzt.First, 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. After denaturation by heating, 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.
Die drei Verfahrensschritte, nämlich Hitzedenaturierung, Primerannealing und Polymerisation, können solange wiederholt werden, bis der Ansatz erschöpft ist. Da bei jedem Einzelschritt eine Verdopplung der DNA-Menge stattfindet, erreicht man nach etwa 20 Zyklen theoretisch einen Vermehrungsfaktor von etwa 10 .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.
Bei der vorliegenden Erfindung werden als Primerpaare solche aus der fimA- Sequenz mit der Bezeichnung Muta 1 und 2 (Abb. 1 ) und solche aus der focA-Sequenz mit der Bezeichnung Muta 3 und 4 (Abb. 2) des Stammes DSM 6601 eingesetzt. Diese DNA-Sequenzen weisen eine teilweise Übereinstimmung mit Genen anderer Enterobakterien auf, aber andererseits gibt es an einigen Positionen Basen, die dort bei anderen Enterobakterien bisher nicht beobachtet werden konnten.In the present invention, 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.
Die weiteren Primerpaare Muta 5 und 6 (Abb. 3), Muta 7 und 8 sowie Muta 9 und 10 (Abb. 4) wurden aus den DNA-Sequenzen der Plasmi pMUT 1 (Abb. 5) und pMUT 2 (Abb. 6) des Stammes DSM 6601 ausgewählt. Diese Primerpaare weisen ebenfalls eine Nucleotidsequenz auf, die bisher bei Enterobakterien so nicht vorgefunden wurde. Die Sequenzen der Primer Muta 1 bis Muta 10 sind in den beigefügten Abbildungen 1 bis 4 im Detail dargestellt.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.
Die Erfindung wird im folgenden anhand des Beispiels näher erläutert:The invention is explained in more detail below using the example:
Eine Kolonie von E. coli Stamm DSM 6601 wird von einer Agarplatte abgeimpft und in 100 μl bidesti liiertem Wasser suspendiert. Diese Suspension wird 10 min auf 95° C erwärmt und anschließend auf Eis abgekühlt. 1 μ\ der Bakteriensuspension wird als Template-DNA für die PCR verwendet.A colony of E. coli strain DSM 6601 is inoculated from an agar plate and suspended in 100 μl bidistilled water. This suspension is heated to 95 ° C. for 10 minutes and then cooled on ice. 1 μ \ of the bacterial suspension is used as template DNA for the PCR.
Daran anschließend wird folgender PCR-Reaktionsansatz in ein PCR- Reaktionsgefäß einpipettiert:The following PCR reaction mixture is then pipetted into a PCR reaction tube:
28 μl H2Obidest 10 μl 5x PCR-Puffer 8 JE/I 1 ,25 mM dNTPs je 1 μl Primer (0,5 μg/μl) 1 μl Template28 μl H 2 O bidest 10 μl 5x PCR buffer 8 JE / I 1, 25 mM dNTPs each 1 μl primer (0.5 μg / μl) 1 μl template
1 μl Taq-Polymerase (1 U/μl)1 μl Taq polymerase (1 U / μl)
Für die PCR-Reaktion wurden folgende Bedingungen gewählt:The following conditions were selected for the PCR reaction:
a. 3 min 95° C (Denaturieren) b. 45 s bei 95° C (Denaturieren) c. 45 s bei 58° C (Annealing der Primer) d. 45 s bei 72° C (Reaktionstemperatur der Taq-Polymerase)a. 3 min 95 ° C (denaturing) b. 45 s at 95 ° C (denaturing) c. 45 s at 58 ° C (annealing of the primers) d. 45 s at 72 ° C (reaction temperature of the Taq polymerase)
Die Schritte b. bis d. werden mindestens 20 mal wiederholt.The steps b. to d. are repeated at least 20 times.
Die Endprodukte können dann beispielsweise zur Identifizierung von Escherichia coli Stamm DSM 6601 angewendet werden oder auch in an sich bekannter Weise sequenziert und zur Überprüfung von entsprechend hergestellten DNA-Sequenzen aus zu untersuchenden E.-coli-Stämmen eingesetzt werden. 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.

Claims

Patentansprüche claims
1. Verfahren zur Identifizierung von Escherichia coli Stamm DSM 6601 , dadurch gekennzeichnet, daß in einer PCR-Reaktion die Primerpaare Muta 1 bis Muta 10 mit den in den Abbildungen 1 bis 4 dargestellten1. A method for identifying Escherichia coli strain DSM 6601, characterized in that in a PCR reaction the primer pairs Muta 1 to Muta 10 with those shown in Figures 1 to 4
DNA-Sequenzen eingesetzt werden.DNA sequences are used.
2. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß die PCR- Reaktion bei folgenden Temperaturen durchgeführt wird:2. The method according to claim 1, characterized in that the PCR reaction is carried out at the following temperatures:
a) 3 min bei 95° C b) 45 s bei 95° C c) 45 s bei 58° C d) 45 s bei 72° Ca) 3 min at 95 ° C b) 45 s at 95 ° C c) 45 s at 58 ° C d) 45 s at 72 ° C
3. Verfahren nach Anspruch 1 und 2, dadurch gekennzeichnet, daß die Schritte b) bis d) mindestens 20 mal wiederholt werden.3. The method according to claim 1 and 2, characterized in that steps b) to d) are repeated at least 20 times.
4. DNA-Sequenzen mit oder aus den in Abbildung 1 dargestellten Nucleotidfolgen.4. DNA sequences with or from the nucleotide sequences shown in Figure 1.
5. DNA-Sequenzen mit oder aus den in Abbildung 2 dargestellten Nucleotidfolgen.5. DNA sequences with or from the nucleotide sequences shown in Figure 2.
6. DNA-Sequenzen mit oder aus den in Abbildung 3 dargestellten Nucleotidfolgen. DNA-Sequenzen mit oder aus den in Abbildung 4 dargestellten Nucleotidfolgen. 6. DNA sequences with or from the nucleotide sequences shown in Figure 3. DNA sequences with or from the nucleotide sequences shown in Figure 4.
EP98966241A 1997-11-19 1998-11-18 METHOD FOR IDENTIFYING $i(ESCHERICHIA COLI) STRAIN DSM 6601 Withdrawn EP1038035A1 (en)

Applications Claiming Priority (3)

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DE19751243 1997-11-19
DE19751243 1997-11-19
PCT/EP1998/007398 WO1999025870A1 (en) 1997-11-19 1998-11-18 Method for identifying escherichia coli strain dsm 6601

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CZ (1) CZ289739B6 (en)
EE (1) EE200000231A (en)
HU (1) HUP0004409A3 (en)
NO (1) NO20002550L (en)
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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN109576198A (en) * 2018-11-09 2019-04-05 南京工业大学 One plant of recombination bacillus coli for knocking out fimA gene and its construction method and application

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DE19915772C2 (en) * 1998-11-18 2001-08-30 Pharma Zentrale Gmbh Method for the identification of Escherichia coli strain 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 (en) * 2001-11-15 2003-06-12 Degussa recA negative and RhaB negative microorganism
CN109843310A (en) 2016-08-31 2019-06-04 哈佛学院院长等 Secrete engineering bacterium and its application method of human cytokines
US20230287438A1 (en) * 2020-09-15 2023-09-14 Northeastern University Plasmid vectors for in vivo selection-free use with the probiotic e. coli nissle

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DE19713543B4 (en) * 1997-04-02 2007-01-11 Pharma-Zentrale Gmbh Bacterial plasmids
DE19751242C2 (en) 1997-11-19 2001-02-08 Pharma Zentrale Gmbh DNA sequences from Fimbrien genes from Escherichia coli strain DSM 6601

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Title
See references of WO9925870A1 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109576198A (en) * 2018-11-09 2019-04-05 南京工业大学 One plant of recombination bacillus coli for knocking out fimA gene and its construction method and application
CN109576198B (en) * 2018-11-09 2022-06-07 南京工业大学 Recombinant escherichia coli with fimA gene knocked out, and construction method and application thereof

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US6489107B1 (en) 2002-12-03
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JP2004516801A (en) 2004-06-10
NO20002550L (en) 2000-07-18
CZ20001873A3 (en) 2000-10-11
EE200000231A (en) 2001-06-15
WO1999025870A1 (en) 1999-05-27
CZ289739B6 (en) 2002-03-13
NO20002550D0 (en) 2000-05-18
HUP0004409A3 (en) 2001-09-28
PL190847B1 (en) 2006-02-28

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