WO1996012038A1 - Procede de controle de bacteries formant des endospores - Google Patents

Procede de controle de bacteries formant des endospores Download PDF

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Publication number
WO1996012038A1
WO1996012038A1 PCT/GB1995/002418 GB9502418W WO9612038A1 WO 1996012038 A1 WO1996012038 A1 WO 1996012038A1 GB 9502418 W GB9502418 W GB 9502418W WO 9612038 A1 WO9612038 A1 WO 9612038A1
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WO
WIPO (PCT)
Prior art keywords
coding region
conserved regions
endospore
dna
sample
Prior art date
Application number
PCT/GB1995/002418
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English (en)
Inventor
Michael Young
Original Assignee
University Of Wales, Aberystwyth
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 GB9420774A external-priority patent/GB9420774D0/en
Application filed by University Of Wales, Aberystwyth filed Critical University Of Wales, Aberystwyth
Priority to EP95933543A priority Critical patent/EP0786013A1/fr
Priority to AU36163/95A priority patent/AU3616395A/en
Publication of WO1996012038A1 publication Critical patent/WO1996012038A1/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

Definitions

  • the present invention relates to a method of monitoring endospore-forming bacteria.
  • Endospore-forming bacteria are important contaminants of food. Some of these bacteria are highly virulent pathogens, such as Clostridium botulinum and Clostridi m perfringens . Bacterial spores are difficult to eliminate because they are resistant to heating and many other sterilising treatments. They can be introduced at almost any stage during food manufacture in raw materials or as a result of careless handling, lack of hygiene or the like. Constant monitoring of raw materials, food production plants, factories or the like is therefore necessary to guard against gross contamination of food by these pathogens.
  • the spoOA gene (or related homologs) is believed to occur in many Gram-positive endospore-forming bacteria such as Bacillus and Clostridium and to be absent from all non endospore- forming bacteria.
  • the SpoOA protein product of the gene is a member of the response regulator super-family of ••two-component" signal transduction proteins.
  • the SpoOA protein is an ambiactive transcription factor which is activated by phosphorylation to stimulate or repress expression of specific sets of target genes.
  • the phosphorylated form of the SpoOA protein directly controls initiation of endospore formation and also influences, typically via an indirect mechanism, other stationary phase phenomena in endospore-forming organisms.
  • the SpoOA protein has highly conserved regions within its carboxy and amino terminal domains, which domains are connected by a linker region, which region varies in sequence and length depending on the organism.
  • deg ⁇ (or a related homolog) concerned with regulation of extracellular enzyme production in endospore-forming bacteria has also been found to have a conserved two-domain structure connected by a variable linker region. It has also been found that gene organisation in endospore-forming bacteria is substantially conserved. The highly conserved genes are connected by intergenic linker (non coding) regions of variable length, depending on the organism.
  • a method of monitoring endospore-forming bacteria present in a sample comprises the steps of adding to the sample primers which are specific for conserved regions in a coding region in the genome of said bacteria, which coding region comprises two domains with a linker region connecting the two domains and which coding region is characteristic of an endospore-forming bacterium, carrying out a DNA amplification procedure so as to amplify conserved regions of the coding region present in said sample; and analysing for the conserved regions.
  • DNA amplification technology it is possible by using DNA amplification technology to monitor the presence of endospore-forming bacteria which might be present in a given sample. The presence of DNA corresponding to the conserved regions is indicative of the presence of endospore-forming bacteria.
  • the conserved regions comprise segments of the amino and carboxy terminal domains of a single gene, such as ⁇ poOA or deg ⁇ , the conserved regions being linked by the variable linker region.
  • the conserved regions of the coding region comprise segments of separate adjacent genes such as spoIVB and spoOA whose disposition in the genome of the endospore-forming bacteria is highly conserved, and which genes are connected by a variable intergenic linker region.
  • a method of identifying endospore-forming bacteria comprises the steps of adding primers to a sample containing endospore-forming bacteria, said primers being specific for the conserved regions of a coding region in the genome of the bacteria, which coding region is characteristic of an endospore-forming bacterium, carrying out a DNA amplification step so as to amplify conserved regions of the coding region and analysing the amplified DNA obtained, wherein said primers are such that they simultaneously amplify a DNA linker region of variable length connecting the conserved regions of the coding region, the length of the DNA fragment obtained from said amplification step being characteristic of a particular endospore-forming bacterium.
  • endospore-forming bacteria which may be present in a given sample may be identified, based on the length of the variable DNA fragment connecting the conserved regions of the coding region, the linker region being amplified by the primers in addition to the conserved regions.
  • the conserved regions of the coding region comprise segments of the amino and carboxy terminal domains of a single gene, such as spoOA or deg ⁇ , the conserved regions being separated by the variable linker region.
  • the conserved regions comprise segments of separate adjacent genes whose disposition in the genome of the endospore-forming bacteria is highly conserved and which genes and their disposition in the genome are characteristic of a particular endospore-forming bacterium, the genes being connected by an intergenic non-coding region.
  • the amplification step comprises the polymerase chain reaction, isothermal amplification or the like.
  • the sample may be a clinical specimen, foodstuff or the like.
  • a pair Of primers TIAARCCITTYGA and TAICCYTTIATRTGIGCIGGIACICC complementary to residues 107-111 (LKPFD) and 168-174 (PAHIKGY - reverse) of the deduced amino acid sequence of spoOA from B . subtilis were used to amplify DNA segments from several bacilli and clostridia using a Perkin Elmer GeneAmpTM PCR System 2400.
  • Each reaction tube contained 100 pmole of each primer, 0.5 ⁇ g of target DNA, all four dNTPs at 200 ⁇ M each, 50mM KCI, 2mM MgCl 2 , lOmM Tris/HCl, 0.001% gelatin (Sigma), pH 8.0, and 2.5U of AmpliTaq DNA polymerase (Perkin Elmer Cetus) .
  • After denaturation for 5 minutes at 94°C samples were amplified for 35 cycles comprising a 40 second denaturation period at 94°C, a 40 second annealing period at 40°C and a 40 second extension period at 72°C. Amplified products were analysed by electrophoresis through a 1.5% agarose gel. The results obtained are given in Table 1.
  • Bacillus stearothermophilus 185 Bacillus stearothermophilus 185
  • the DNA fragments were transferred by standard capillary blotting methods to a nylon membrane (Hybond N, Amersham) and hybridized under stringent conditions with 24-mer oligonucleotides (see below) that had been labelled at their 3' ends with digoxigenin, using a commercially available 3'-end labelling kit (Boehringer Mannheim) .
  • the oligonucleotide specifically detected the PCR product derived from the organism from which the primer had been synthesized; none of the PCR products derived from any of the other organisms were detected.
  • the sequences of the oligonucleotides employed in this experiment, together with the sequences of the corresponding region of the spoOA linker region, were as indicated in Table 2.
  • a pair of primers ATIGARTGGATGYT and TAICCYTTIATRTGIGCIGGIACICC, complementary to residues 407-411 (IEWML) of ⁇ poIVB from B . subtilis and 168-174 (PAHIKGY - reverse) of spoOA from B . subtil is were used to amplify DNA segments from several bacilli and clostridia using a Perkin Elmer GeneAmpTMPCR System 2400.
  • Each reaction tube contained 100 pmole of each primer, 0.5 ⁇ g of target DNA, all four dNTPs at 200 ⁇ M each, 50mM KCI, 2mM MgCl 2 , lOmM Tris/HCl, 0.01% gelatin (Sigma), pH 8.0, and 2.5 U of AmpliTaq DNA polymerase (Perkin Elmer Cetus). After denaturation for 5 min at 94°C samples were amplified for 35 cycles comprising a 40s denaturation period at 94°C, a 40s annealing period at 40°C and a 40s extension period at 72°C. Amplified products were analysed by electrophoresis through a 1.5% agarose gel. The results obtained are given in Table 3.

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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

On détecte ou on identifie des bactéries formant des endospores présentes dans un échantillon par apport audit échantillon d'amorces spécifiques à des régions conservées dans une région codante du génome des bactéries et par exécution d'un processus d'amplification d'ADN. Dans le procédé de détection, on peut amplifier toute région conservée présente dans l'échantillon et effectuer ensuite une analyse de détermination de la présence des régions conservées. Dans le procédé d'identification, on amplifie les régions conservées de la région codante et on analyse ensuite l'ADN amplifié obtenu, les amorces étant de nature à amplifier simultanément une séquence codante variable de l'ADN intermédiaire aux régions conservées de la région codante, la longueur du fragment d'ADN obtenu dans l'étape d'amplification étant caractéristique d'une bactérie particulière formant des endospores.
PCT/GB1995/002418 1994-10-14 1995-10-12 Procede de controle de bacteries formant des endospores WO1996012038A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP95933543A EP0786013A1 (fr) 1994-10-14 1995-10-12 Procede de controle de bacteries formant des endospores
AU36163/95A AU3616395A (en) 1994-10-14 1995-10-12 Method of monitoring endospore-forming bacteria

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB9420774A GB9420774D0 (en) 1994-10-14 1994-10-14 Method of detecting and identifying spore forming bacteria
GB9420774.3 1994-10-14
GB9518661A GB2300481B (en) 1994-10-14 1995-09-13 Monitoring endospore-forming bacteria involving the amplification of DNA of conserved regions thereof
GB9518661.5 1995-09-13

Publications (1)

Publication Number Publication Date
WO1996012038A1 true WO1996012038A1 (fr) 1996-04-25

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1995/002418 WO1996012038A1 (fr) 1994-10-14 1995-10-12 Procede de controle de bacteries formant des endospores

Country Status (3)

Country Link
EP (1) EP0786013A1 (fr)
AU (1) AU3616395A (fr)
WO (1) WO1996012038A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19801661A1 (de) * 1998-01-17 1999-07-22 Artus Ges Fuer Molekularbiolog Schnell-Detektionsverfahren für Organismen durch Längenbestimmung ausgewählter Nukleinsäurebereiche
WO1999061667A1 (fr) * 1998-05-27 1999-12-02 Betzdearborn Inc. Amorces pour la detection de bacteries sporulees dans des produits de papiers et courants de systemes de fabrication de papier
WO2002092853A1 (fr) * 2001-05-15 2002-11-21 Hercules Incorporated Detection de bacteries formant des spores

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0409159A2 (fr) * 1989-07-18 1991-01-23 Shimadzu Corporation Procédé d'essai de microorganismes à l'origine d'intoxication alimentaire et réactifs utilisés dans ce but
EP0502271A1 (fr) * 1989-04-17 1992-09-09 The Standard Oil Company Sondes d'oligonucléotides de 16s rDNA pour l'identification de bactéries sulfato-réductrices

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0502271A1 (fr) * 1989-04-17 1992-09-09 The Standard Oil Company Sondes d'oligonucléotides de 16s rDNA pour l'identification de bactéries sulfato-réductrices
EP0409159A2 (fr) * 1989-07-18 1991-01-23 Shimadzu Corporation Procédé d'essai de microorganismes à l'origine d'intoxication alimentaire et réactifs utilisés dans ce but

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BROWN, P. ET AL: "Characterization of spoOA homologues in diverse Bacillus and Clostridium species identifies a probable DNA-binding domain", MOLECULAR MICROBIOLOGY, vol. 14, no. 3, pages 411 - 26 *
VAN HOY, B. ET AL: "Characterization of the spoIVB and recN loci of Bacillus subtilis", JOURNAL OF BACTERIOLOGY, vol. 172, no. 3, pages 1306 - 11 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19801661A1 (de) * 1998-01-17 1999-07-22 Artus Ges Fuer Molekularbiolog Schnell-Detektionsverfahren für Organismen durch Längenbestimmung ausgewählter Nukleinsäurebereiche
WO1999061667A1 (fr) * 1998-05-27 1999-12-02 Betzdearborn Inc. Amorces pour la detection de bacteries sporulees dans des produits de papiers et courants de systemes de fabrication de papier
WO2002092853A1 (fr) * 2001-05-15 2002-11-21 Hercules Incorporated Detection de bacteries formant des spores

Also Published As

Publication number Publication date
EP0786013A1 (fr) 1997-07-30
AU3616395A (en) 1996-05-06

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