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WO2004101817A1 - Identification et detection de campylobacter - Google Patents

Identification et detection de campylobacter Download PDF

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Publication number
WO2004101817A1
WO2004101817A1 PCT/AU2004/000654 AU2004000654W WO2004101817A1 WO 2004101817 A1 WO2004101817 A1 WO 2004101817A1 AU 2004000654 W AU2004000654 W AU 2004000654W WO 2004101817 A1 WO2004101817 A1 WO 2004101817A1
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Prior art keywords
campylobacter
seq
sample
nucleic acid
profile
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Inventor
Feng Shi
Benjamin Nicholas Fry
Peter John Coloe
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Microtechnology Centre Management Ltd
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Microtechnology Centre Management Ltd
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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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to the identification of bacterial infection or detection of contamination, in particular, by means of genotyping.
  • the present invention is directed to providing a diagnostic test which may be carried out to detect the presence of, and optionally identify the species, strain or type of Campylobacter in a sample, in particular in a sample from a patient, particularly a human patient, enabling prompt and accurate diagnosis and hence effective treatment with appropriate drugs.
  • the diagnostic test of the present invention is a simple and rapid test, which is convenient and quick to perform.
  • Campylobacter jejuni and Campylobacter coli are recognised as one of the most common causes of food-borne bacterial gastroenteritis. Furthermore, Campylobacter jejuni has been implicated as a frequent antecedent to the development of the neurologic diseases Guillain-Barre syndrome (GBS; Mishu and Blaser, 1993) and Miller Fisher syndrome (MFS; Salloway et al. , 1996).
  • LPS lipopolysaccharide
  • LPS is a major constituent of the outer membrane in Gram -negative bacteria and comprises three covalently linked regions: Lipid A, core oligosaccharide (inner core and outer core) and O polysaccharide. LPS from Campylobacter was found to lack O polysaccharide in most strains. It is more like lipooligosaccharide (LOS) in H. influenzae and N. meningitidis .
  • LOS lipooligosaccharide
  • Campylobacter LOS outer core and capsule polysaccharide The variability of the Campylobacter LOS outer core and capsule polysaccharide is thought to contribute to the antigenic basis of the Penner serotyping system. Serotyping methods like these are time consuming, technically demanding and antisera are costly to produce, which limits these typing systems to specialised diagnostic laboratories. Furthermore, phenotypes can be unstable resulting in non-reproducible results or non- typeable strains.
  • Genotyping methods are independent of expressed features and are therefore a better alternative.
  • Several genotyping methods have recently been developed, such as pulsed- field gel electrophoresis (PFGE; Imai et al, 1994; Yan et al, 1991; Gibson et al, 1995).
  • PFGE pulsed- field gel electrophoresis
  • MMT Mutilocus sequence typing
  • AFLP Amplified fragment length polymorphism
  • PCR- RFLP Aim et al, 1993; Ayling et al, 1996; ⁇ achamkin et al, 1993, 1996; Owen et al, 1993b
  • ribotyping ribotyping; Fayos et al, 1992; Owen et al, 1993a; Russell et al, 1994
  • RAPD random amplified polymorphic D ⁇ A
  • genotyping systems are more generally available and applicable than phenotypic methods. However, most of these techniques still have their own drawbacks such as limited discriminatory abilities, complex techniques and poor reproducibility. More importantly, none of these methods correlate well with the serotyping scheme used in the past decades, so that historical ep.idemiological trends cannot be pursued. For these reasons, genotypic subtyping methods have not been widely used in epidemiological practice.
  • a LOS biosynthesis gene cluster of Campylobacter jejuni 81116 has been characterised (Fig. 1 ; Fry et al, 1998) and sequenced. A PCR-RFLP typing scheme has been established based on this LOS cluster (Shi et ah, 2002). A second LOS gene cluster was sequenced from some C. jejuni strains (Gilbert et al, 2000, 2002) and more strains are currently being sequenced. The inventors of the present application have identified a means of utilising the LOS gene cluster in a method for diagnosing Campylobacter infection and identifying the genotype of a sample.
  • One aspect of the present invention provides a method for identifying Campylobacter in a sample comprising the steps of : (i) performing nucleic acid amplification by contacting the sample with a pair of nucleic acid primers, wherein at least one nucleic acid primer is derived from the wla gene cluster of Campylobacter, for a time and under conditions sufficient for generation of an amplification product; (ii) preparing a profile of the amplification product; and
  • the present invention further comprises prior to step (ii), the step of : (iv) digesting the sample or amplification product with a restriction endonuclease enzyme
  • the present invention provides a method of identifying Campylobacter in a sample comprising the steps of : (i) performing nucleic acid amplification by contacting the sample with a pair of nucleic acid primers, for a time and under conditions sufficient for generation of an amplification product comprising the wla gene cluster of Campylobacter or a portion thereof; (ii) preparing a profile of the amplification product; and (iii) comparing the profile to a profile of an comparator Campylobacter species, strain or type, and thereby determining the identity of Campylobacter in the sample.
  • the present invention further comprises the step of digesting the sample or amplification product with a restriction endonuclease enzyme prior to step (ii).
  • the present invention provides a method for identifying
  • Campylobacter in a sample comprising the steps of:
  • the invention provides a kit for performing the above methods.
  • the kit comprises:
  • nucleic acid primers (a) a pair of nucleic acid primers, wherein at least one nucleic acid primer is derived from the wla gene cluster of Campylobacter; or (b) a pair of nucleic acid primers for amplification of the wla gene cluster of
  • Campylobacter or a portion thereof or (c) a probe capable of hybridising to the wla gene cluster of Campylobacter.
  • the kit further comprises one or more reaction buffers suitable for use in a nucleic acid hybridisation reaction (Southern-Blot or Microarray) or polymerase chain reaction.
  • kit further comprises at least one restriction endonuclease enzyme.
  • Figure 1 is a representation of the physical and genetic map of the wla gene cluster of C. jejuni. The restriction sites are shown.
  • Figure 2 is two sequence alignments of the waaC (Fig. 2A) and waaF (Fig. 2B) gene from different C. jejuni strains.
  • Figure 3 is photographic representation showing PCR amplification products from C. jejuni strains separated by electrophoresis on an agarose gel and visualised with ethidium bromide.
  • the PCR primers used were WaaC ⁇ TTGGTGCAACACCAAGCTACCGCAAT (SEQ ID NO:l)
  • PCR amplification products were separated on a 0.8 % (w/v) agarose gel.
  • Lane 1 is strain 81116
  • lane 2 is HS-1
  • lane 3 is HS-2
  • lane 4 is HS-5
  • lane 5 is HS-8
  • lane 6 is HS-13
  • M is ⁇ DNA digested with Pstl
  • lane 7 is HS-19
  • lane 8 is HS-22
  • lane 9 is HS- 44
  • lane 10 is HS-50 and lane 11 is HB9313.
  • the profiles of the PCR products varied from 9kb to 14kb.
  • Figure 4 is a photographic representation of RFLP profiles of 11 C. jejuni strains after PCR, separated by electrophoresis on a 1.5% agarose gel and visualised with ethidium bromide.
  • the PCR primers used were
  • WaaC6 TTGGTGCAACACCAAGCTACCGCAAT (SEQ ID NO: 1)
  • H ⁇ II was used to digest the PCR products.
  • Lane 1 is strain 81116
  • lane 2 is ⁇ S-1
  • lane 3 is HS-2
  • lane 4 is HS-5
  • lane 5 is HS-8
  • lane 6 is 13
  • M is ⁇ DNA digested with Pstl
  • lane 7 is HS-19
  • lane 8 is HS-22
  • lane 9 is HS-44
  • lane 10 is HS-50 and lane 11 is HB9313.
  • Figure 5 A is a photographic representation of an oligo array hybridised with HS-23 5kb target PCR product.
  • the PCR primers used were
  • OrfS-1 GGCCCTGATATAGTTTGTTCTATGGAT (SEQ ID NO:20)
  • the oligo array is a 12x15 dot array. Each dot represents a particular oligonucleotide (oligo). Each oligo has a duplicate spot next to it. Each array has a duplicate block on the slide. Three different oligo lengths were tested for the best performance under the optimum hybridisation conditions.
  • Figure 5B is a diagram of the names and positions of the oligos used in the oligonucleotide array depicted in Figure 5 A.
  • Figure 6 is the sequence of the wla gene cluster of strain ATCC 43469.
  • the inventors have discovered that the molecular composition, physical arrangements and nucleotide sequences of the wla gene cluster are different in different types of Campylobacter.
  • the inventors have utilised the diversity in the design of reliable diagnostic assays for the typing of Campylobacter.
  • the diagnostic assays described herein provide a significant advantage over currently employed assays based upon the detection, of Campylobacter DNA and provide results which correspond with conventional serotyping of Campylobacter spp.
  • one aspect of the present invention provides a method for identifying Campylobacter in a sample by performing nucleic acid amplification using a pair of nucleic acid primers wherein at least one nucleic acid primer is derived from the wla gene cluster of Campylobacter, or a pair of nucleic acid primers which amplify the wla gene cluster, to produce an amplification product.
  • the profile of the amplification product is compared to same from an comparator Campylobacter, wherein a similarity indicates the identity of Campylobacter in the sample.
  • the present invention provides a method for identifying Campylobacter in a sample comprising digesting the sample with at least one restriction endonuclease enzyme and detecting the hybridisation of a probe to the wla gene cluster of Campylobacter to the sample.
  • the resultant profile is compared with a profile of a comparator Campylobacter, to determine the identity of Campylobacter in the sample.
  • the term “wla gene cluster” refers to a region or part thereof of the
  • Campylobacter genome comprising a cluster of genes involved in lipooligosaccharide biosynthesis (sometimes referred to as lipopolysaccharide biosynthesis).
  • a "part" of the wla gene cluster comprises at least 10, more preferably at least 15, 20, 30, 40, 50 or 100 contiguous nucleotides of the wla gene cluster.
  • the wla gene cluster may include, but is not limited to the following genes, their homologues, analogues and derivatives (Fig.
  • N-acetylglucosamine-6-phosphatase (SEQ ID NO: 15) neuAl CMP-Neu5Ac synthetase (SEQ ID NO: 16) orfl 1 putative glycosyltransferase (SEQ ID NO : 17) waaV putative glucosyltransferase (SEQ ID NO: 18) waaF ADP-heptose-LPS heptosyltransferase (SEQ ID NO: 19)
  • probe refers to a nucleic acid molecule which is derived from a Campylobacter spp. and capable of being used in the detection of the wla gene cluster.
  • primer refers to a probe as hereinbefore defined which is further capable of being used to amplify a Campylobacter wla gene cluster in a polymerase chain reaction.
  • the probe or primer or a homologue, analogue or derivative thereof is derived from C. jejuni and C. coli, amongst others.
  • the primer or probe is derived from the wla gene cluster or an adjacent region.
  • An "adjacent region" is a region that is sufficiently close to the wla gene cluster that it may be amplified or detected by primers or probes within the wla gene cluster.
  • the primers or probe are derived from the more conserved regions within the wla gene cluster. More preferably primers can be designed to PCR waaC-waaF fragment from all C. jejuni strains.
  • the genotyping system of the present invention was developed based on genetic variations of the DNA region in the wla cluster.
  • the waaC gene in the wla cluster was shown to be conserved by gene distribution analysis from 20 different C. jejuni Penner serotype strains.
  • the waaC and waaF genes are a particularly suitable target for a genotyping system.
  • the primer or probe is derived from waaC and or waaF or a complementary nucleotide sequence or a homologue, analogue or derivative thereof.
  • there is a pair of nucleic acid primers wherein one primer is derived from the wla gene cluster of Campylobacter.
  • the other primer of the pair may be derived from the wla gene cluster of Campylobacter or an adjacent region. Where the other primer is derived from an adjacent region, it must be sufficiently close to the wla gene cluster that the use of the pair of primers is capable of amplifying the intervening region.
  • the probe or primer is at least 80%, 90% or 95% identical to one or more of the sequences set forth in SEQ ID Nos: 1-6, 20-21 (Table 1) or a complementary nucleotide sequence, or a homologue, analogue or derivative thereof.
  • the probe or primer is capable of hybridising under high stringency conditions to one or more of the sequences set forth in SEQ ID NOS: 1-6, 20-21 or to a complementary nucleotide sequence or a homologue, analogue or derivative thereof.
  • the probe or primer used in the invention preferably comprises a sequence of nucleotides of at least 15 nucleotides, more preferably at least 25 nucleotides, even more preferably at least 50 nucleotides and even more preferably at least 100 nucleotides or 500 nucleotides derived from the sequence set forth in SEQ ID NOs:l-6 and 20-21 or a complement thereof.
  • the probe or primer comprises a nucleotide sequence set forth in any one or more of SEQ ID NOS: 1-6, 20-21 or a complementary nucleotide sequence, or a homologue, analogue or derivative thereof.
  • the primers are hybridised to nucleic acids of Campylobacter in the sample being analysed, as probe pairs, in the combinations comprising SEQ ID Nos: 1-6 and 20-21; or complementary strands, homologues, analogues or derivatives thereof.
  • the present invention particularly contemplates the use of primers as set forth in any one or more of SEQ ID Nos: 1-6 and 20-21 as being useful in the identifying Campylobacter species as well as for detecting Campylobacter in a sample.
  • nucleotide sequence shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as the nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence within said sequence, of one or more nucleotide substitutions, insertions, deletions, or rearrangements.
  • nucleotide sequence set forth herein shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as a nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence of any non-nucleotide constituents not normally present in said isolated nucleic acid molecule, for example carbohydrates, radiochemicals including radio nucleotides, reporter molecules such as, but not limited to biotin, DIG, alkaline phosphatase or horseradish peroxidase, amongst others.
  • references to "derivatives" of a nucleotide sequence or “derived from” a nucleotide sequence as set forth herein shall be taken to refer to any isolated nucleic acid molecule which contains significant sequence similarity to said sequence or a part thereof.
  • nucleotide sequence of the present invention may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or insertions.
  • Nucleotide insertional derivatives of the nucleotide sequence of the present invention include 5 ' and 3 ' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides or nucleotide analogues.
  • Insertional nucleotide sequence variants are those in which one or more nucleotides or nucleotide analogues are introduced into a predetermined site in the nucleotide sequence of said sequence, although random insertion is also possible with suitable screening of the resulting product being performed.
  • Deletional variants are characterised by the removal of one or more nucleotides from the nucleotide sequence.
  • Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide or nucleotide analogue inserted in its place.
  • the present invention encompasses all such homologues, analogues or derivatives, subject to the proviso that said homologues, analogues or derivatives are useful in the performance of at least one assay format as described herein.
  • the probe or primer may comprise inosine, adenine, guanine, thymidine, cytidine or uracil residues or functional analogues or derivatives thereof which are capable of being incorporated into a polynucleotide molecule, provided that the resulting probe or primer is capable of hybridising under at least low stringency conditions to a Campylobacter genome.
  • a low stringency is defined herein as being a hybridisation and/or a wash carried out in 6xSSC buffer, 0.1% (w/v) SDS at 28°C.
  • a moderate stringency is defined herein as being a hybridisation and/or wash carried out in 2xSSC buffer, 0.1% (w/v) SDS at a temperature in the range 45°C to 65°C.
  • a high stringency is defined herein as being a hybridisation and/or wash carried out in O.lxSSC buffer, 0.1% (w/v) SDS at a temperature of at least 65°C.
  • the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash.
  • the conditions for hybridisation and/or wash may vary depending upon the nature of the hybridisation membrane or the type of hybridisation probe used. Conditions for hybridisations and washes are well understood by one normally skilled in the art. For the purposes of clarification of the parameters affecting hybridisation between nucleic acid molecules, reference is found in pages 2.10.8 to 2.10.16. of Ausubel et al (1987), which is herein incorporated by reference.
  • the probe may be labelled with a reporter molecule capable of producing an identifiable signal (e.g. a radioisotope such as 32 P or 35 S or a biotinylated molecule).
  • a reporter molecule capable of producing an identifiable signal
  • the detection of said reporter molecule provides for detection of the Campylobacter probe and that, following hybridisation, the detection of the corresponding Campylobacter wla gene cluster in the biological sample is facilitated.
  • additional probes may be used to confirm the assay results obtained using a single probe.
  • nucleic acid amplification refers to any of a range of amplification reactions including PCR, nucleic acid sequence-based amplification (NASBA) system, rolling circle amplification and Q ⁇ replicase based amplification amongst them.
  • NASBA nucleic acid sequence-based amplification
  • the amplification is by PCR or modifications thereof.
  • one or more nucleic acid primer molecules of at least 15 contiguous nucleotides is hybridised to a template molecule comprising nucleic acid derived from said sample and nucleic acid copies of the wla gene cluster or portions thereof present in said sample or a part or fragment thereof are enzymically-amplified.
  • a template molecule comprising nucleic acid derived from said sample and nucleic acid copies of the wla gene cluster or portions thereof present in said sample or a part or fragment thereof are enzymically-amplified.
  • the hybridisation conditions may be varied to promote hybridisation.
  • the primer is at least 95% identical to the complement of the nucleotide sequence in the template molecule to which it hybridises.
  • the polymerase chain reaction provides for the hybridisation of non-complementary primers to different strands of a template molecule, such that the hybridised primers are positioned to facilitate the 5N6 3N synthesis of nucleic acid in the intervening region, under the control of a thermostable DNA polymerase enzyme.
  • the polymerase chain reaction provides an advantage in so far as the nucleotide sequence in the region between the hybridised primers may be unknown and unrelated to any known nucleotide sequence.
  • amplification product shall be understood to refer to a nucleic acid molecule, which is the result of or has been subjected to amplification.
  • a “profile” means the relative or approximate or absolute length, molecular weight, isoelectric charge or size of a nucleic acid molecule whether single stranded, double stranded or a combination thereof.
  • a profile may be the pattern of bands or spots produced by the detection of nucleic acid on a solid phase or the data obtained therefrom.
  • the profile serves to identify the Campylobacter species, strain or type in a sample by comparison to a comparator Campylobacter, wherein a similar profile indicates the identity of Campylobacter in the sample as that of the comparator Campylobacter.
  • the species, strain or type of the comparator Campylobacter is known. In the event it is unknown, the Campylobacter in the sample is identified as being of the same or similar type to the comparator Campylobacter.
  • the profile of a nucleic acid molecule can be detected by any nucleic acid detection means and more specifically, a nucleic acid sequence specific detection means or non-sequence specific detection means.
  • Non-sequence specific detection means include autoradiography, fluorescent dyes, and silver staining. These methods are well known to the skilled person.
  • Sequence specific detection means include, for example sequencing, nucleic acid hybridisation techniques or paper chromatography hybridisation assay (PACHA) as described by Reinhartz et al. (1993) and equivalents thereof. Additional variations of this detection means, which utilise the nucleotide sequences described herein, are clearly encompassed by the present invention.
  • the primers are selected such that, when nucleic acid derived from the biological sample, in particular DNA, is amplified, different profiles are produced from different Campylobacter ssp. and/or types.
  • the amplification means described supra may be further coupled to a classical hybridisation reaction and detection means to further enhance sensitivity and specificity of the inventive method, in particular by hybridising the amplified DNA with a probe which is different from any of the primers used in the amplification reaction.
  • the amplification products may be subjected to electrophoresis, transferred to a solid support such as, for example, a nylon or nitrocellulose membrane, and hybridised to the probe as hereinbefore defined, optionally labelled with a reporter molecule.
  • a specific amplified DNA fragment is hybridised to the probe, and a profile detected which is specific for a particular Campylobacter ssp. and/or types to enable the user to distinguish between different species or types in much the same way as for RFLP analysis.
  • a particularly preferred embodiment of the inventive method comprises the step of detecting the amplified nucleic acid by contacting one or more of the nucleotide sequences set forth in SEQ ID Nos: 1-2 thereto for a time and under conditions sufficient for hybridisation to occur.
  • hybridisation means described supra may be further coupled to a second hybridisation step employing a probe which is different from the probe used in the first hybridisation reaction.
  • nucleic acid derived from the biological sample, in particular DNA is digested with one or more restriction endonuclease enzymes.
  • the enzymes may be chosen based on an analysis of the sequence of known strains.
  • Suitable restriction endonuclease enzymes include, but are not limited to, Hhal, Ddel, Nlalll, Hindlll, Clal, Bglll, Xbal, Spel, Xhol, EcoR ⁇ , Rsal and particularly Hhal, Rsal, Ddel, Nlalll and Hindlll.
  • the digested DNA may be subjected to electrophoresis to separate the components.
  • a solid support such as, for example, a nylon or nitrocellulose membrane, and hybridised to the probe as hereinbefore defined, optionally labelled with a reporter molecule.
  • a specific profile of DNA fragments is hybridised to the probe, and the profile is specific for a particular Campylobacter ssp. and/or type, to enable the user to distinguish between different species and/or types.
  • the Campylobacter being identified is selected from the list comprising C. jejuni and in particular types ⁇ S-3, HS-7, HS-8, HS-13, HS-22, HS-50, C.coli and in particular types HS-5.
  • the method is used to identify the type of Campylobacter jejuni present in a sample. More preferably the typing accords with traditional serotyping. For example, the typing identifies a Campylobacter jejuni strain as belonging to a Penner serotype. Penner serotypes are HS-1, HS-2, HS-3, HS-5, HS-6, HS-7, HS-8, HS-13, HS-19, HS-22, and HS-50.
  • the sample may be a biological sample obtained from a human or animal.
  • the sample may be a food or food ingredient sample or a sample derived from a utensil or appliance used in the preparation, storage or serving of food.
  • a sample may be prepared in a suitable solution, for example an extraction buffer or suspension buffer.
  • food encompasses any substance for human or animal consumption, whether nutritional or medicinal.
  • Other samples sources include shellfish, raw chicken and raw milk as well as healthy cattle and flies.
  • Non-chlorinated water may also be a sample source.
  • the sample may be derived from human stool, blood or vomit.
  • clinical isolates on agar slants can be suspended in 10 mM EDTA in a microcentrifuge tube. The tube is boiled for five minutes and the resultant lysate, contains the nucleic acid for use in the present invention.
  • the nucleic acid may comprise a crude or partially-pure extract of Campylobacter DNA or RNA or alternatively, comprise amplified DNA or purified wla gene cluster DNA.
  • the invention may further comprise a method of quantifying Campylobacter content in a sample.
  • Competitive polymerase chain reaction is a standard method for quantifying DNA.
  • Real-time PCR using the TaqMan (registered trade mark) system developed by PE Biosystems (Foster City, CA, USA) allows rapid detection and quantification of DNA without the need for labour intensive post-PCR processing such as gel electrophoresis and radioactive hybridisation (6).
  • the built-in 96 well format greatly increases the number of samples, which can be simultaneously analysed.
  • the method uses the 5' exonuclease activity of a Taq polymerase (AmpliTaq Gold, PE Biosystems, Foster City, CA, USA) during primer extension to cleave a dual-labelled, fiuorogenic probe hybridised to the target DNA between the PCR primers.
  • a reporter dye such as 6-carboxyfluorescein (6-FAM) at the 5' end of the probe is quenched by 6-carboxy-tetramethylrhodamine (TAMRA) through fluorescent resonance energy transfer.
  • TAM 6-carboxy-tetramethylrhodamine
  • the invention provides a kit for conducting the above methods.
  • the kit comprises
  • nucleic acid primers (a) a pair of nucleic acid primers, wherein at least one nucleic acid primer is derived from the wla gene cluster of Campylobacter; or
  • kits capable of hybridising to the wla gene cluster of Campylobacter.
  • the kit of the present invention may be compartmentalised to contain in a first compartment, one or more probes or primers are hereinbefore defined.
  • the invention extends further to such kits wherein both primers of a primer pair are provided in the same compartment, in aqueous solution or dried, such that the subject primers are at a relative concentration suitable for subsequent use in an amplification reaction.
  • the invention clearly extends to kits at least comprising one or more pairs of said primers.
  • the kit optionally comprises several further containers comprising a reaction buffer suitable for use in one or more of the detection means described herein and optionally several third containers comprising a nucleic acid molecule positive standard, to which the profile may be compared.
  • a negative control reaction is carried out in which the contents of the first container are contacted with the contents of the second container.
  • the sample to be tested is contacted with the contents of the first and second containers for a time and under conditions sufficient for hybridisation to occur. If the reagents contained in the first container provided are not labelled with a reporter molecule, then the contents of the first container may be so labelled prior to the hybridisation reaction being carried out.
  • the hybridised test sample and the negative control sample are then subjected to a detecting means as hereinbefore described. In analysing the profile obtained using said kit, the control negative control reaction, test sample and nucleic acid molecule positive standard are compared side-by-side.
  • the contents of the third container should always provide a positive result upon which to compare the profile obtained for the negative control and test sample. If the test sample produces a profile, which is substantially the same as that contained in the positive standard, albeit of different intensity, then the sample contains a Campylobacter ssp. of the same type as the positive standard .
  • Penner serotypes of Campylobacter spp. were detected using the Passive haemagglutination (PHA) technique as described by Penner and Hennessy (Penner and Hennessy, 1980), with a heated supernatant from the bacterial culture as antigen.
  • Antisera were prepared by injecting rabbits intravenously with saline bacterial suspensions of Campylobacter serostrains. Some antisera were absorbed with cross reacting HS reference strains to clarify serotyping results, in particular antiserum prepared against HS4 was absorbed with HS16 bacteria to remove minor reactions with HS13 and HS16.
  • DNA was isolated from pure cultures by the cetyltrimethylammonium (CTAB) procedure (Ausubel et al, 1995). Briefly, one lawn plate of Campylobacter was grown overnight, harvested in 9.4ml TE buffer and 0.1ml 0.5 M EDTA and lysed with 0.5ml 10% (w/v) SDS. Proteinase K was added to a final concentration of 0.1 mg/ml and the mixture was incubated at 37 ° C for 2h. Then 1.8ml 5M NaCI and 1.5ml 10% (w/v) CTAB in 0.7M NaCI were added and the mixture was incubated for 30 min at 65 ° C.
  • CTAB cetyltrimethylammonium
  • PCR was performed in a 50 ⁇ l reaction volume using a Perkin Elmer GeneAMP 2400 thermal cycler.
  • the reaction mixtures consisted of lx reaction buffer with 2.0 mM MgSO 4 (Promega), 2.5 units Taq DNA polymerase (Promega), 100 ng forward and reverse primer, 0.2 mM dNTP's and 100 ng template DNA.
  • the reaction included an initial denaturation of DNA at 94 °C for 1 minute and then 35 cycles of consecutive denaturation (30 seconds, 94 °C), primer annealing (30 seconds, 60 °C), and chain extension (based on a rate of 1 kb/minute, 72 ° C).
  • a final elongation step was 10 minutes at 72 ° C.
  • the w ⁇ C and waaF genes were detected in the multiple C. jejuni strains investigated, indicating both genes are highly conserved among C. jejuni serotype reference strains and a suitable source of a primer for amplification of the wla gene cluster.
  • WaaC4 CGCTTGGGGAAATGTAAAAGAATACGAAT - (SEQ ID NO:4)
  • WaaC6 TTGGTGCAACACCAAGCTACCGCAAT (SEQ ID NO: 1)
  • WaaF3 TAAGGJC04CTTTACCAAGTTCTTTGCG Sail (SEQ ID NO:6)
  • WaaF5 TTGCATACAAGGCATACAAGCTAGAT (SEQ IDNO:2)
  • Bold italicised nucleotides indicate the restriction sites in the primers; Underlined nucleotides indicate the modifications from original sequence of the wla cluster of C. jejuni 81116.
  • the primers WaaC6 (SEQ ID NO:l) and WaaF5 (SEQ ID NO:2) were used to amplify fragment between the w ⁇ C and w ⁇ F genes of C. jejuni strains 81116, HS-1, HS-5, HS-8, HS-44 and HB9313.
  • PCR was performed by using Expand Long Template PCR system (Roche).
  • the reaction mixtures consisted of lx reaction buffer 1 with 1.75 mM MgCl 2 , 2.65 units polymerase enzyme mix, 100 ng forward and reverse primer, 0.35 mM dNTP's and 100 ng template DNA.
  • the reaction included an initial denaturation of DNA at 93 °C for 2 minutes and then 35 cycles of consecutive denaturation (10 seconds, 93 °C), primer annealing (30 seconds, 61 °C), and chain extension (10 minutes, 68 ° C, cycle elongation for more yield of 20 seconds for each cycle after 10 cycles).
  • a final elongation step was 7 minutes at 68 ° C.
  • the primer pair WaaC6 (SEQ ID NO: l)-WaaF5 (SEQ ID NO:2) gave PCR products for 7 out of 11 C. jejuni Penner -serotype reference strains.
  • the profiles of the PCR products varied from 9kb to 14kb (Fig. 3).
  • Hindlll was chosen to digest the obtained amplification product from 11 C. jejuni strains. The enzymes were chosen based on an analysis of the sequence of known strains.
  • 10 ⁇ l PCR product was digested with 10 units of restriction enzymes (Hindlll, Promega) in a total volume of 20 ⁇ l with 2 ⁇ g BSA for more than 3 hours at 37 °C. The digest was analysed by electrophoresis using a 1.5% agarose gel and stained with ethidium bromide. Lambda DNA digested with Pstl was used as a marker.
  • Eleven strains could by typed by the profile of the PCR amplification product using primers WaaC6 and WaaF5 and seven more easily by their PCR-RFLP profile just using H dill. These types could be correlated with the serotypes in Example 2.
  • Campylobacter LOS sequences were aligned and analysed by using Clone Manager
  • PCR products were used as a template for incorporation of biotin by randomly primed polymerisation reaction. Briefly, PCR products were purified by the Wizard PCR Preps DNA purification system (Promega) and eluted in 35 ⁇ l mili-Q water. Fifteen microliters purified PCR products were heated to 95°C and snap cooled on ice to denature.
  • the labelled target DNA was 95°C heat denatured and snap cooled on ice.
  • the hybridisation mixture consisted of 5 ⁇ l of denatured target DNA and 5 ⁇ l hybridisation buffer (5xSSPE, 0.01% triton X100).
  • the hybridisation mixture was transferred onto epoxy glass slides and covered with a cover slip.
  • the slides were placed in a hybridisation chamber and hybridised in 45°C water bath for 30 minutes. After hybridisation, the cover slips were removed and slides were washed with 2xSSC/0.1% SDS, 0.5xSSC/0.1% SDS, 2xSSC and 4xSSC/0.2%Tween 20 at 45°C. All the washes were performed for 3-5 min with agitated shaking.
  • the slides were then hybridised with
  • Microarray 12x15 and 16x15 sequence (5'-3'Y GC % location differentiation
  • OAl 1-0 CCTATAGTTAACCATAATCCA 33 orfl 1 conserved region represents class A, B
  • thermotolerant species of Campylobacter with ribosomal RNA gene pattern Res. Microbiol. 144:709-720.
  • Ribosomal RNA patterns identify additional strains of Campylobacter jejuni and C. coli among isolates serotyped by heat-stable and heat-labile antigens. Laboratory Animal Sci. 44-579-583.

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Abstract

L'invention concerne l'identification d'une infection bactérienne ou la détection d'une contamination, notamment par génotypage. Elle porte sur la fourniture d'un test de diagnostic pouvant être effectué pour la détection de la présence et éventuellement l'identification de l'espèce, de la souche ou du type de Campylobacter dans un échantillon, notamment dans un échantillon prélevé sur un patient, notamment un patient humain, permettant un diagnostic rapide et précis et donc un traitement efficace avec des médicaments appropriés. Le test de diagnostic de l'invention constitue un test simple et rapide, pratique et facile à mettre en oeuvre.
PCT/AU2004/000654 2003-05-19 2004-05-19 Identification et detection de campylobacter Ceased WO2004101817A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003020958A2 (fr) * 2001-09-04 2003-03-13 Exponential Biotherapies Inc. Typage de campylobacter

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003020958A2 (fr) * 2001-09-04 2003-03-13 Exponential Biotherapies Inc. Typage de campylobacter

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FRY B.N. ET AL.: "The galE gene Campylobacter jejuni is involved in lipopolysaccharide synthesis and virulence", INFECTION AND IMMUNITY, 2000, pages 2594 - 2601 *
FRY B.N. ET AL.: "The lipopolysaccharide biosynthesis locus of Campylbacter jejuni 81116", MICROBIOLOGY, vol. 144, 1998, pages 2049 - 2061 *
KLENA J.D. ET AL.: "Cloning, sequencing, and characterization of the lipopolysaccharide biosynthetic enzyme heptosyltransferase I gene (waaC) from Campylobacter jejuni and Campylobacter coli", GENE, vol. 222, 1998, pages 177 - 185 *
NAOAKI MISAWA T AL.: "DNA diversity of the wla gene cluster among serotype HS:19 and non-HS:19 Campylobacter jejuni strains", JOURNAL OF ENDOTOXIN RESEARCH, vol. 7, no. 5, 2001, pages 349 - 358 *

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