EP0786013A1 - Method of monitoring endospore-forming bacteria - Google Patents

Method of monitoring endospore-forming bacteria

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Publication number
EP0786013A1
EP0786013A1 EP95933543A EP95933543A EP0786013A1 EP 0786013 A1 EP0786013 A1 EP 0786013A1 EP 95933543 A EP95933543 A EP 95933543A EP 95933543 A EP95933543 A EP 95933543A EP 0786013 A1 EP0786013 A1 EP 0786013A1
Authority
EP
European Patent Office
Prior art keywords
coding region
conserved regions
endospore
dna
sample
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.)
Ceased
Application number
EP95933543A
Other languages
German (de)
French (fr)
Inventor
Michael Young
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.)
Aberystwyth University
Original Assignee
Aberystwyth University
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 Aberystwyth University filed Critical Aberystwyth University
Publication of EP0786013A1 publication Critical patent/EP0786013A1/en
Ceased legal-status Critical Current

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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 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

Endospore-forming bacteria present in a sample are detected or identified by adding to the sample primers which are specific for conserved regions in a coding region in the genome of the bacteria, and carrying out a DNA amplification procedure. In the detection method, any of the conserved regions present in the sample may be amplified, followed by analysing for the presence of the conserved regions. In the identification method, the conserved regions of the coding region are amplified, followed by analysing the amplified DNA obtained, the primers being such that they simultaneously amplify a variable coding sequence of DNA intermediate the conserved regions of the coding region, the length of the DNA fragment obtained from the amplification step being characteristic of a particular endospore-forming bacterium.

Description

Method of Monitoring Endospore-Forming Bacteria
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. In particular, 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.
Likewise, another gene, degϋ (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.
We have found that these conserved regions provide an effective marker for monitoring endospore-forming bacteria and that the variable linker regions separating the conserved regions provides highly specific markers for identifying individual species of endospore-forming bacteria.
Accordingly, there is provided by one aspect of the present invention, a method of monitoring endospore-forming bacteria present in a sample, which method 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. Thus, advantageously, 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.
In one embodiment of the invention, the conserved regions comprise segments of the amino and carboxy terminal domains of a single gene, such as εpoOA or degϋ, the conserved regions being linked by the variable linker region. In an alternative embodiment of the invention, 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.
According to a second aspect of the present invention, there is provided a method of identifying endospore-forming bacteria, which method 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.
Thus, advantageously, 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.
In one embodiment, the conserved regions of the coding region comprise segments of the amino and carboxy terminal domains of a single gene, such as spoOA or degϋ, the conserved regions being separated by the variable linker region. Alternatively, 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.
Preferably, the amplification step comprises the polymerase chain reaction, isothermal amplification or the like. Thus, very small numbers of organisms present in the sample may be identified. The sample may be a clinical specimen, foodstuff or the like.
The invention may be more clearly understood with reference to the following examples. Example l
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 GeneAmp™ PCR System 2400. Each reaction tube contained 100 pmole of each primer, 0.5μg of target DNA, all four dNTPs at 200μM each, 50mM KCI, 2mM MgCl2, 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.
Tab] Le l
Organism Approximate Size of Amplified DNA Fragment
Bacillus subtilis 205
Bacillus cereus 195
Bacillus stearothermophilus 185
Bacillus megateriu 190
Bacillus brevis 180
Clostridium beijerinckii 215
Clostridium butyricum 230
Clostridium pasteurianum 205
Clostridium thermoaceticum 170
Clostridium innocuum 205 Exam le 2
Three of the species listed in Table 1, viz Bacillus subtilis, Clostridium pasteurianum and Clostridium innocuu gave PCR products of indistinguishable size. They were differentiated by their ability to hybridize with oligonucleotides corresponding to unique segments of the variable linker region of spoOA.
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) . In each case, 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.
Table 2 organism Peptide Oligonucleotide
B . subtilis NASSVTHR AATGCCAGGAGTGTGACGCATCGT
C. pasteurianum KAADVKIS AAAGCTGCAGATGTAAAGATTTCT
C. innocuum EHISSNVL GAGCATATATCCAGCAATGTGCTT
Example 3
Amplification of variable length products across the intergenic region separating spolΥB and spoOA.
A pair of primers ATIGARTGGATGYT and TAICCYTTIATRTGIGCIGGIACICC, complementary to residues 407-411 (IEWML) of εpoIVB 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 GeneAmp™PCR System 2400. Each reaction tube contained 100 pmole of each primer, 0.5μg of target DNA, all four dNTPs at 200μM each, 50mM KCI, 2mM MgCl2, 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.

Claims

CLAIMS :
1. A method of monitoring endospore-forming bacteria present in a sample, which method comprises the steps of adding to said sample primers which are specific for conserved regions in a coding region in the genome of said bacteria, which coding region comprising 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.
2. A method according to claim 1, wherein said coding region comprises an individual gene, said conserved regions comprising the carboxy and amino terminal domains of said gene.
3. A method according to claim 2 wherein said gene comprises spoOA.
4. A method according to claim 1, wherein said coding region comprises segments of at least two adjacent genes, each of said genes coding for a distinguishable product.
5. A method according to claim 4, where said adjacent genes comprise spoTVB and spoOA.
6. A method of identifying endospore-forming bacteria which 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 said bacteria which coding region has a two- domain structure and 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, the length of the DNA fragment obtained from said amplification step being characteristic of a particular endospore-forming bacterium.
7. A method according to claim 6, wherein said coding region comprises an individual gene, said conserved regions comprising the carboxy and amino terminal domains of said gene.
8. A method according to claim 7, wherein said gene comprises spoOA .
9. A method according to claim 6, wherein said coding region comprises segments of at least two separate adjacent genes coding for distinguishable products.
10 A method according to claim 9, wherein said adjacent genes comprise spoIVB and spoOA
11. A method according to any preceding claim wherein said amplification step comprises polymerase chain reaction amplification or isothermal amplification.
EP95933543A 1994-10-14 1995-10-12 Method of monitoring endospore-forming bacteria Ceased EP0786013A1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GB9420774 1994-10-14
GB9420774A GB9420774D0 (en) 1994-10-14 1994-10-14 Method of detecting and identifying spore forming bacteria
GB9518661A GB2300481B (en) 1994-10-14 1995-09-13 Monitoring endospore-forming bacteria involving the amplification of DNA of conserved regions thereof
GB9518661 1995-09-13
PCT/GB1995/002418 WO1996012038A1 (en) 1994-10-14 1995-10-12 Method of monitoring endospore-forming bacteria

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EP0786013A1 true EP0786013A1 (en) 1997-07-30

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Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19801661A1 (en) * 1998-01-17 1999-07-22 Artus Ges Fuer Molekularbiolog Rapid detection of organisms from analysis of ubiquitous nucleic acid sequences, e.g. for identifying pathogens
US5928875A (en) * 1998-05-27 1999-07-27 Betzdearborn Inc. Primers for the detection of spore forming bacteria
CA2443441A1 (en) * 2001-05-15 2002-11-21 Hercules Incorporated Detection of spore forming bacteria

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0502271A1 (en) * 1989-04-17 1992-09-09 The Standard Oil Company 16s rRNA oligonucleotide probes for the identification of sulfate-reducing bacteria
EP0409159B1 (en) * 1989-07-18 1998-11-25 Shimadzu Corporation Method for testing causative microorganism of food poisoning and reagents therefor

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9612038A1 *

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AU3616395A (en) 1996-05-06

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