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WO2016005767A1 - Détection de micro-organismes viables - Google Patents

Détection de micro-organismes viables Download PDF

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Publication number
WO2016005767A1
WO2016005767A1 PCT/GB2015/052005 GB2015052005W WO2016005767A1 WO 2016005767 A1 WO2016005767 A1 WO 2016005767A1 GB 2015052005 W GB2015052005 W GB 2015052005W WO 2016005767 A1 WO2016005767 A1 WO 2016005767A1
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sample
strand
polymerase
activity
nucleic acid
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PCT/GB2015/052005
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English (en)
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Matthew Alun Crow
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Momentum Bioscience Limited
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Publication of WO2016005767A1 publication Critical patent/WO2016005767A1/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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • 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
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/10—Nucleotidyl transfering
    • C12Q2521/101—DNA polymerase
    • 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
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/50—Other enzymatic activities
    • C12Q2521/501—Ligase
    • 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
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/50—Other enzymatic activities
    • C12Q2521/531—Glycosylase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91245—Nucleotidyltransferases (2.7.7)

Definitions

  • the present invention relates generally to the field of detecting the absence or presence of microorganisms in a sample.
  • the methods typically rely upon measuring microbial enzyme activity (if any) present in a sample and may relate to such methods which are capable of being performed using nucleic acid amplification techniques such as the polymerase chain reaction.
  • the methods of the invention therefore enable determination of the absence and presence of microbial pathogens in samples such as un-purified blood, blood culture and other body fluids.
  • This invention in particular relates to substrate nucleic acid molecules which permit ligase and polymerase activity to be determined and discriminated from one another.
  • the invention also relates to improvements in work flow relating to blood culture sampling to assist with integration of the microorganism detection methods into laboratory testing protocols.
  • Measuring the presence and levels of certain molecules which are associated with cell viability is important in a number of contexts. For example, measuring levels of ATP is useful in mammalian cells for growth analysis and toxicology purposes. Culture approaches can be used to detect small numbers of bacteria but such techniques require several days to complete, especially when attempting to detect small numbers of bacteria and also when detecting slower growing microorganisms.
  • WO96/002665 describes a method for determining the presence and/or amount of microorganisms and/or their intracellular material present in a sample characterized in that the amount of adenylate kinase in the sample is estimated by mixing it with adenosine diphosphate (ADP), determining the amount of adenosine triphosphate (ATP) produced by the sample from this ADP, and relating the amount of ATP so produced to the presence/or amount of adenylate kinase and to microorganisms and/or their intracellular material, wherein the conversion of ADP to ATP is carried out in the presence of magnesium ions at a molar concentration sufficient to allow maximal conversion of ADP to ATP.
  • ADP adenosine diphosphate
  • ATP adenosine triphosphate
  • ligases in particular NAD-dependent ligases, are disclosed as a useful indicator of the presence of a (viable) microorganism in a sample.
  • Ligases are enzymes which catalyze ligation of nucleic acid molecules. The ligation reaction requires either ATP or NAD+ as co-factor depending upon the ligase concerned.
  • the use of NAD-dependent ligase activity is utilized as an indicator of the presence of a (viable) microorganism in a sample.
  • WO201 1/130584 describes a method for detection of viable microorganisms based on detection of DNA or RNA polymerases in which a sample is contacted with a nucleic acid substrate that acts as a substrate for microbial polymerase, incubated under conditions suitable for polymerase activity from intact microorganisms and any resulting nucleic acid product is determined using a nucleic acid amplification technique such as quantitative polymerase chain reaction.
  • a nucleic acid amplification technique such as quantitative polymerase chain reaction.
  • the ETGA assay is unable to distinguish microorganism polymerase activity from that of the host or from dead microorganisms.
  • Applicant's co-pending application WO2010/1 19270 describes a method for removing enzyme activity (in this case, DNA ligase) outside intact microorganisms and this can be used also for removal of contamination nucleic acid polymerase activity.
  • enzyme activity in this case, DNA ligase
  • determining their absence the applicants do not mean that the sample is necessarily sterile but may have an organism load that is sufficiently low as to be negative for practical purposes.
  • blood cultures are often taken from patients suspected of having bloodstream infections which can be associated with sepsis, a condition that can be rapidly fatal if left untreated. It is routine for clinical microbiology laboratories to incubate such specimens for at least five days before reporting a negative result, during which time the patients are often kept on broad spectrum antibiotics. Typically up to 90% of such patients are negative, and so a large number of patients are left for 5 days on antibiotic therapy that is not necessary for their condition.
  • a faster method for determining negative growth would be of significant value in reducing the cost of unnecessary antibiotic therapy and provide health benefits in terms of reducing the risks of C. difficile infection, antibiotic toxicity and antimicrobial resistance. Identifying negative cultures requires the ability to detect both bacterial and fungal/yeast infections if present.
  • the foundation of the invention is methods of detecting the absence or presence of a micro-organism in a sample comprising:
  • the invention is based on developments in existing ETGA assays such as those assays relying upon detection of polymerase and/or ligase activity.
  • yeast a potentially fatal source of bloodstream infection (if left untreated) display poor polymerase activity in ETGA assays. Accordingly, there is benefit in being able to perform assays in which both ligase and polymerase activity is detected.
  • the inventors have devised a substrate nucleic acid molecule that permits both ligase and polymerase activity to be determined and, importantly, discriminated.
  • the invention provides a nucleic acid substrate for ligase and/or polymerase activity comprising:
  • a second strand c. a complementary strand that is complementary to the first and second strands such that the first and second strands hybridize with the complementary strand to produce a substantially double stranded structure
  • first strand acts as a primer for polymerase activity and the complementary strand acts as a template strand to thus extend the first strand in the presence of polymerase activity
  • the 5' end of the second strand is phosphorylated to permit ligation of the 3' end of the first strand and the 5' end of the second strand in the presence of ligase activity wherein the second strand contains a sequence that does not base pair with the complementary strand sequence such that there is a mismatched region in the substantially double stranded structure
  • the complementary strand is sensitive to treatment to render it no longer a template strand, whereas the first and second strands are resistant to such treatment.
  • a suitable structure is shown schematically in Fig. 1 .
  • the first and second strands when hybridized with the complementary strand form a largely continuous strand. The two are not joined however, the 3' end of the first strand is not (covalently) bonded to the 5' end of the second strand. This is important to permit ligation and/or extension reactions to take place if the appropriate enzyme activity is present in the sample.
  • the substrate may be extended by polymerase activity. All bacteria and fungi express DNA and RNA polymerase activity. The present invention does not rely upon discrimination of the source of polymerase activity.
  • the first strand of the nucleic acid substrate acts as a primer for polymerase activity and the complementary strand acts as a template strand to thus extend the first strand in the presence of polymerase activity. This extension reaction effectively displaces the second strand which is downstream in the 5'-3' direction of the first strand.
  • the substrate may be ligated by bacterial or fungal ligase.
  • Bacterial ligase is a DNA ligase which depends upon the nicotinamide adenine dinucleotide (NAD+) cofactor for activity.
  • NAD-dependent ligase It is referred to hereinafter as "NAD-dependent ligase”.
  • fungal ligases are ATP-dependent ligases which rely upon the cofactor adenosine triphosphate (ATP) for activity. They are referred to hereinafter as “ATP-dependent ligase”.
  • ATP-dependent ligase The activity of both types of ligase is the formation of a phosphodiester bond between the 5' end of a nucleic acid molecule and the 3' end of a nucleic acid molecule.
  • the 5' end of the second strand is the formation of a phosphodiester bond between the 5' end of a nucleic acid molecule and the 3' end of a nucleic acid molecule.
  • the complementary strand is sensitive to treatment to render it no longer a template strand, whereas the first and second strands are resistant to such treatment.
  • the complementary strand may be modified in a manner such that it is sensitive to treatment to render it no longer a template strand.
  • the other parts of the nucleic acid substrate i.e. the first and second strands
  • This is important to permit discrimination of polymerase and ligase activity in a downstream amplification process. This ensures that, during a subsequent amplification reaction (a step in ETGA assays), the polymerase used in the amplification reaction is unable to amplify the complementary strand using the first strand as a primer.
  • the complementary strand of the nucleic acid substrate comprises uracil bases/nucleotides such that upon treatment with uracil DNA glycosylase (UDG) enzyme the complementary strand is no longer able to serve as a template strand.
  • the first and second strands do not contain uracil bases/nucleotides.
  • the complementary strand may incorporate heat labile nucleotides such that prior to, or during, the subsequent amplification process, a heating step may be employed in order to (selectively) degrade the complementary strand. This prevents amplification of the complementary strand using the first strand as a primer in the amplification reaction. No heating step is employed in the initial detection method to permit extension and/or ligation.
  • the first and second strands may be modified such that they are resistant to a treatment applied to the complementary strand.
  • the first and second strands may be modified such that they are resistant to enzymatic, such as nuclease, degradation.
  • Any suitable means may be employed in order to protect the first and second strands from nuclease activity.
  • Non-limiting examples include incorporation of methylation into the nucleic acid molecule, end modification such as protection of the 3' and/or 5' ends and incorporation of synthetic nucleotides.
  • the synthetic nucleotides comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides.
  • the synthetic nucleotides replace at least one up to all of the nucleotides in the nucleic acid molecule.
  • end modification is sufficient to protect from exonuclease activity.
  • the complementary strand is preferentially degraded. This prevents that strand from being amplified in the subsequent detection step.
  • the first and second strands, as part of a ligated or extended product remain amplifiable as they are resistant to the exonuclease treatment.
  • the modifications to the first and second strand should not preclude or inhibit the ligation or extension reactions.
  • the second strand contains a (portion of) sequence that does not base pair with the complementary strand sequence such that there is a mismatched region in the substantially double stranded structure. This is key to being able to subsequently discriminate those nucleic acid molecules that have been ligated versus those that have been extended by polymerase. In the presence of polymerase activity, the second strand is displaced and so a sequence complementary to the sequence of the
  • complementary strand is produced.
  • the complementary (template) strand is copied thus displacing the second strand from the substantially double stranded structure.
  • the second strand is ligated to the first strand and so the region of mismatch is retained.
  • the region of potential mismatch can be probed to determine the source of the enzyme activity in the sample (ligase and/or polymerase). The mismatched region can be positioned anywhere within the second strand.
  • the mismatched region is not found within 1 , 2, 3, 4, 5, 10, 15, 20, 25 or 30 nucleotides of one or both ends of the second strand, in particular the 5' end (to avoid any impact on ligation efficiency).
  • the mismatched region can be of any length provided that the mismatch is detectable and does not adversely affect the functioning of the assay (i.e. the extension or ligation reactions).
  • the mismatch should not prevent overall hybridization between the first and second strand respectively and the complementary strand, even though the region of mismatch will not hybridize.
  • the mismatch is a short region (relative to the overall length of the molecule).
  • the mismatched region is between (and including) 3-50, such as 5-25 or 6-12 nucleotides in length.
  • the mismatched region may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 nucleotides in length in some embodiments.
  • the mismatched region is flanked by significant regions of identity between the second strand and the complementary strand.
  • the regions of identity may be at least 10, 15, 20, 30, 40, 50, 100 or more nucleotides in length.
  • the nucleic acid substrates may be provided in either orientation, according to standard nomenclature.
  • the first and second strands are sense strands and the complementary strand is an antisense strand.
  • the first and second strands are antisense strands and the complementary strand is a sense strand.
  • the nucleic acid substrates may be DNA based. They may incorporate synthetic nucleotide analogues as appropriate or may be RNA or PNA based for example, or mixtures thereof. Suitable modifications, for example, to protect the substrate from nuclease activity, may be included. Any suitable means may be employed in order to protect the nucleic acid molecule from nuclease activity. Non-limiting examples include incorporation of methylation into the nucleic acid molecule, end modification such as protection of the 3' and/or 5' ends and incorporation of synthetic nucleotides.
  • the synthetic nucleotides comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides. In certain embodiments, the synthetic nucleotides replace at least one up to all of the nucleotides in the nucleic acid molecule. As is discussed above, modifications may preferentially be made to certain components of the substrate in specific embodiments.
  • the nucleic acid substrates may be labelled, such as using a fluorescent label, or FRET pair, in certain embodiments to facilitate detection. Suitable detection methods are described herein.
  • the substrate nucleic acid molecules include any natural nucleic acid and natural or synthetic analogues that are capable of being acted upon by nucleic acid modifying activity in order to generate the extended and/or ligated nucleic acid molecule.
  • the substrate may be extended and/or ligated in specific embodiments.
  • the nucleic acid substrate is present in excess, and in particular in large molar excess, over the nucleic acid modifying activity (provided by the microorganisms) in the sample. This is an important technical distinction over prior art methods. Because a novel extended and/or ligated nucleic acid molecule is detected, only the presence of this molecule in the sample is essential for the detection methods to work effectively. Thus, it is not detrimental to the methods of the invention if other nucleic acid molecules are present in the sample such as from the microorganisms to be detected or from mammalian or other sources which may be found in the sample to be tested for example.
  • the substrate and/or primers may incorporate complementary non-naturally occurring molecules which can base pair with each other, to avoid nonspecific detection of genomic DNA.
  • pyDAD and puADA may be incorporated into primers and substrate molecules as appropriate (Sismour et al., Nucleic Acids Research, 2004, Vol. 32, No. 2: 728-735).
  • the invention provides for use of a nucleic acid substrate as described herein for detecting polymerase and/or ligase activity in a sample. Specific methods are described herein.
  • the invention further provides a kit for detecting the presence or absence of polymerase and/or ligase activity in a sample comprising:
  • nucleic acid substrate as defined herein
  • a first primer which hybridizes to the product of ligation between the first and
  • a second primer which hybridizes to the amplification product resulting from use of the first primer and directs further amplification of that amplification product; and/or f. uracil DNA glycosylase and/or a nuclease enzyme such as an exonuclease enzyme (depending upon the form of substrate used).
  • the kit may include a probe which hybridizes to a target sequence comprising at least a portion of the mismatched region contained within the second strand. This enables ligated molecules, which may subsequently have been amplified, to be detected.
  • the kit may include or further include a probe which hybridizes to a target sequence comprising the sequence corresponding to at least a portion of the mismatched region in the complementary strand. This enables extended molecules, produced as a consequence of polymerase activity (in which the second strand is displaced) to be detected (optionally following an amplification step).
  • the kit may include both probe types.
  • Either one or both probes may be labelled to facilitate detection.
  • Typical labels include chromophores, fluorophores and electrochemically active species. Where both probe types are included in the kits each probe may be differently labelled.
  • By “differently labelled” is meant a detectably different label thus permitting the probes to be easily distinguished from one another (e.g. using one detection methodology).
  • the skilled person would be readily able to select suitable different labels.
  • the labels may have different wavelengths of maximal emission.
  • Suitable pairs of fluorophores which may be utilised include Cy3 and FAM or Texas Red and FAM.
  • Other fluorophores which may be employed include Alexa dyes, JOE, TET, HEX etc.
  • the kit may additionally or alternatively incorporate reagents for performing a subsequent amplification step (once the ligated and/or extended nucleic acid substrate has been generated).
  • reagents include a first primer which hybridizes to the product of ligation between the first and second strands and/or to the product of polymerization from the first strand acting as primer and the complementary strand acting as template and directs amplification of the product of ligation/polymerization.
  • the primer hybridizes outside of the mismatch region and thus will amplify extension or ligation products.
  • the first primer may incorporate the probe function such that the primer binds to either extension or ligation products but not both.
  • primers may be labelled and differently labelled if they perform the probe function in relation to identifying the origin of the mismatch sequence.
  • the kit may incorporate a second primer which hybridizes to the amplification product resulting from use of the first primer and directs further amplification of that amplification product.
  • Suitable primer pairs can be designed by one skilled in the art against any defined nucleic acid substrate molecule.
  • the second primer (but typically not both the first and second primer although this may be possible if the mismatch region is large enough) may incorporate the probe function such that the primer binds to either extension or ligation products but not both.
  • two separate second primers may be required to allow amplification of both product types. The discussion of probes above therefore applies mutatis mutandis to these embodiments.
  • primers may be labelled and differently labelled if they perform the probe function in relation to identifying the origin of the mismatch sequence.
  • the complementary strand of the nucleic acid substrate is sensitive to treatment to render it no longer a template strand, whereas the first and second strands are resistant to such treatment.
  • the first and second strands are resistant to such treatment.
  • complementary strand comprises modification such that it is sensitive to treatment to render it no longer a template strand, such as incorporation of uracil bases such that upon treatment with uracil DNA glycosylase (UDG) enzyme the complementary strand is no longer a template strand.
  • first and second strands may be modified to protect them from a treatment, such as (exo)nuclease treatment as discussed herein. This ensures that, during a subsequent amplification reaction (a step in ETGA assays), the polymerase used in the amplification reaction is unable to amplify the complementary strand using the first strand as a primer.
  • the kit incorporates uracil DNA glycosylase.
  • UDG is available from various sources.
  • UDG may be termed UNG and this is the approved gene symbol (according to the HUGO Gene Nomenclature Committee).
  • the enzyme may be recombinantly produced in a suitable expression system (e.g. in E. coli).
  • UDG may be from any origin, e.g. bacterial or human.
  • the human gene reference sequence is NM 08091 1 and the Genbank accession number is A64377. Homologs exist in a number of species including mice and rats. Suppliers include New England Biolabs and Thermo Scientific.
  • the enzyme is typically applied to control carry-over contamination in PCR.
  • the kit contains modified first and second strands and a nuclease, such as an exonuclease.
  • kits of the invention may usefully be applied to discriminate bacteria from fungi in a (clinical) sample. Suitable methods are discussed in further detail herein.
  • the invention may require deactivation of (bacterial) NAD-dependent ligase activity in the sample.
  • the kit of the invention may include reagents for
  • the reagents may be affinity based reagents that permit removal of NAD-dependent ligase activity from the sample or inactivate the NAD-dependent ligase activity within the sample.
  • the reagents may specifically bind to NAD-dependent ligase.
  • Suitable reagents may comprise antibodies, aptamers or peptides. They may be immobilised on a solid support to facilitate rapid removal from the sample. For example they may be immobilised on beads, such as magnetic beads that can be readily separated from the sample. In the context of a kit the solid support may be provided separately optionally together with means for immobilising the specific binding agent on the solid support.
  • the antibody may be of monoclonal or polyclonal origin. Typically, the antibody is an IgG immunoglobulin isotype. Antigen-binding fragments and antibody derivatives may also be utilised, to include without limitation Fab fragments, ScFv, single domain antibodies, nanoantibodies, heavy chain antibodies, chimeric antibody fusions etc. which retain antigen-specific binding function and these are included in the definition of "antibody”. Methods for generating specific antibodies are known to those skilled in the art. Antibodies may be of human or non-human origin (e.g. rodent, such as rat or mouse) and be humanized etc. according to known techniques (Jones et al., Nature (1986) May 29-Jun.
  • the aptamer may be a nucleic acid molecule, to include natural nucleotides and derivatives/analogues and non-natural nucleotides, or a peptide molecule, to include natural amino acids and derivatives/analogues and non-natural amino acids bonded with standard or non-standard peptide bonds, or one or more operably connected nucleic acid molecules and/or peptide molecules.
  • the aptamer is a DNA, RNA or XNA aptamer. Nucleic acid aptamer selection can be made using methods known to those skilled in the art, for example using in vitro selection or SELEX.
  • the aptamer is a peptide aptamer comprising a variable peptide domain, attached at both ends to a protein scaffold.
  • the variable peptide domain may be up to 30 amino acids long, preferably 5 to 25 amino acids long, more preferably 10 to 20 amino acids long.
  • the scaffold may be any protein which has appropriate solubility and compacity properties, for example the bacterial protein Thioredoxin-A, Peptide aptamer selection can be made using methods known to those skilled in the art, for example using the yeast two-hybrid system.
  • an antibody binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope.
  • the term is well known in the art. Accordingly, an antibody specifically binds to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope.
  • a specific binding agent such as an aptamer, protein or peptide specifically binds to a target molecule when it binds to that molecule more readily than it would to a random, unrelated molecule.
  • specificity of binding ensures that NAD-dependent ligase is inactivated/removed but ATP-dependent ligase activity in the sample is not inactivated/removed.
  • kits of the invention chemical reagents for deactivating NAD-dependent ligase activity in the sample may be employed and included in the kits of the invention. Examples include
  • Reducing conditions to reduce NAD and thus prevent it from activating NAD dependent ligase.
  • These reagents may preferentially affect NAD-dependent ligase activity and not significantly affect, or affect to a lesser degree, ATP-dependent ligase activity in the sample.
  • reagents may inhibit NAD-dependent ligase activity where the ligase is not already “charged” with NAD.
  • charge is meant that an AMP group derived from
  • NAD is covalently attached to the NAD-dependent ligase, typically via a conserved lysine residue within the KXDG motif of the NAD-dependent ligase.
  • a dummy or false substrate may be included in the kits (for addition to the sample). This false substrate can be acted upon by the ligase but is not detected and does not impair subsequent detection of the nucleic acid substrate molecule of the invention. Thus, typically the false substrate does not include the primer and/or probe binding sites found in the nucleic acid substrate molecules of the invention.
  • the kits may incorporate both a chemical reagent to inhibit NAD-dependent ligase activity and a false substrate molecule.
  • the kit may incorporate suitable reagents for amplification, such as PCR in particular quantitative PCR (qPCR).
  • PCR quantitative PCR
  • the kit comprises or further comprises one or more of a polymerase, dinucleotide triphosphates, MgCI 2 and buffer.
  • Any suitable polymerase may be utilised. Examples include thermostable polymerases such as Taq or Pfu polymerase and the various derivatives of those enzymes.
  • Suitable buffers are also well known and commercially available and may be included in a PCR mastermix that includes the majority of the components required for PCR amplification.
  • kits of the invention are useful for detecting polymerase and/or ligase activity in a sample.
  • the invention provides corresponding methods using the substrate nucleic acid molecules described herein.
  • a method for detecting polymerase and/or ligase activity in a sample comprising:
  • treating the sample for example by adding UDG to the sample or heating the sample if a substrate is used with a heat-labile complementary strand or adding a nuclease if a protected first and second strand is utilised, in order to render the complementary strand no longer a template strand
  • detecting the amplification products to detect polymerase and/or ligase activity in the sample e. detecting the amplification products to detect polymerase and/or ligase activity in the sample.
  • step e comprises as part of the detection, discriminating amplification products arising from ligase or polymerase activity in the sample. This may be achieved using suitable primers and/or probes which bind to the mismatched region or the sequence corresponding to the mismatched region in the complementary strand.
  • step e comprises the addition of a first probe which hybridizes to a target sequence comprising the mismatched region contained within the second strand together with a second probe which hybridizes to a target sequence comprising the sequence corresponding to the mismatched region in the complementary strand.
  • discriminating amplification products arising from ligase or polymerase activity relies upon detecting hybridization of the probes to their respective target sequences.
  • the probes may be differently labelled for convenience of detection, as discussed in further detail herein.
  • the primers may incorporate the probe functionalities (e.g. if using hairpin primers or hairpin probe containing primers, such as Scorpion or Amplifluor primers) and thus a separate probe is not necessarily required.
  • the method comprises, prior to step a, removing from the sample or deactivating in the sample any NAD-dependent ligase activity.
  • a dummy or false substrate may be utilised in the methods prior to step a.
  • This false substrate can be acted upon by the ligase but is not detected and does not impair subsequent detection of the nucleic acid substrate molecule of the invention.
  • the false substrate does not include the primer and/or probe binding sites found in the nucleic acid substrate molecules of the invention.
  • the methods may require use of both a chemical reagent to inhibit NAD-dependent ligase activity and a false substrate molecule in some embodiments.
  • the invention provides a method of detecting the absence or presence of a microorganism in a sample comprising performing a method as described herein and using the detection of polymerase and/or ligase activity in the sample as an indicator of the absence or presence of a microorganism in the sample.
  • the microorganism may be a bacterium or a fungus (e.g. a yeast).
  • the methods may involve further characterising the nature of the microorganism once detected in the sample.
  • the invention further provides a method for detecting and discriminating bacteria and/or fungi in a sample, comprising:
  • treating the sample for example by adding UDG to the sample or heating the sample if a substrate is used with a heat-labile complementary strand or adding a nuclease if a protected first and second strand is utilised, in order to render the complementary strand no longer a template strand e performing a nucleic acid amplification step in order to amplify any ligated first and second strand in the sample (due to ligase activity) and/or extended first strand in the sample (due to polymerase activity)
  • detecting and discriminating the amplification products to detect polymerase and/or ligase activity in a sample wherein the presence of polymerase activity in the sample but no ligase activity indicates the presence of bacteria in the sample but not fungi and/or wherein the presence of ligase activity in the sample indicates the presence of fungi and/or wherein the presence of both polymerase and ligase activity in the sample indicates the presence of fungi and possibly also bacteria.
  • This method may require, as a preliminary step (depending upon the sample type utilised), removal of non-microorganism (such as mammalian) sources of nucleic acid modifying activity from the sample.
  • non-microorganism such as mammalian
  • This may be achieved for example by lysing non- microorganism cells in the sample as a preliminary step, followed by inactivation and/or removal of the nucleic acid modifying activity provided by the non-microorganism cells.
  • the fact that the microorganisms remain intact protects them from inactivation or removal of their own nucleic acid modifying activity during this step.
  • This may then be followed by a second lysis step to release the microorganism source of nucleic acid modifying activity to be detected.
  • the lysis of microorganisms may be separated into a lysis step for fungi and a subsequent lysis step for bacteria.
  • the subsequent lysis step may be performed in the presence of inhibitors of NAD-dependent ligase (as described herein). This permits ligase activity detected according to the methods to be attributed to fungi only, because (ATP-dependent) ligase activity from the fungi is not impaired by the inhibitors until the bacteria have been lysed and the inhibitors added.
  • the fungal ligase By lysing the fungal cells initially and incubating for a suitable period of time (under suitable conditions), the fungal ligase (if present in the sample) will already have acted on the nucleic acid substrate before the bacterial cells are lysed and the inhibitors added (which may then impact on further activity of the fungal ligases).
  • the lysis of microorganisms may be separated into a lysis step for bacteria followed by removal of the bacterial lysed cell material and a subsequent lysis step for fungi.
  • This may avoid the need to utilise NAD-dependent ligase inhibitors.
  • the bacterial lysis step may be performed in the presence of inhibitors of NAD-dependent ligase (as described herein). This permits ligase activity detected according to the methods to be attributed to fungi only, because (ATP-dependent) ligase activity from the fungi is not impaired by the inhibitors since they are removed with the bacterial lysed cell mixture.
  • the presence of NAD-dependent ligase inhibitors may reduce the risk of carry over contamination into the fungal lysis step (which could potentially give a false positive signal of fungal ligase activity if active NAD- dependent ligase was carried over).
  • Separate lysis steps may be achieved by use of appropriate selective lysis reagents.
  • commercially available reagents include B-Per (Thermo Scientific) for lysis of bacterial cells and Y-Per (Thermo Scientific) for lysis of yeast/fungal cells.
  • Step a may require applying to the sample reagents for deactivating NAD-dependent ligase activity.
  • Any suitable reagents may be employed, as discussed in detail in respect of the kits of the invention.
  • the reagents may be affinity based reagents that permit removal of NAD-dependent ligase activity from the sample or inactivate the activity within the sample.
  • the reagents may specifically bind to NAD-dependent ligase.
  • Suitable reagents may comprise antibodies, aptamers or peptides. They may be immobilised on a solid support to facilitate rapid removal from the sample. For example they may be immobilised on beads, such as magnetic beads that can be readily separated from the sample.
  • the antibody may be of monoclonal or polyclonal origin. Typically, the antibody is an IgG immunoglobulin isotype. Antigen-binding fragments and antibody derivatives may also be utilised, to include without limitation Fab fragments,
  • the aptamer may be a nucleic acid molecule, to include natural nucleotides and derivatives/analogues and non-natural nucleotides, or a peptide molecule, to include natural amino acids and derivatives/analogues and non- natural amino acids bonded with standard or non-standard peptide bonds, or one or more operably connected nucleic acid molecules and/or peptide molecules.
  • the aptamer is a DNA, RNA or XNA aptamer.
  • the aptamer is a peptide aptamer comprising a variable peptide domain, attached at both ends to a protein scaffold.
  • the variable peptide domain may be up to 30 amino acids long, preferably 5 to 25 amino acids long, more preferably 10 to 20 amino acids long.
  • the scaffold may be any protein which has appropriate solubility and compacity properties, for example the bacterial protein Thioredoxin-A.
  • chemical reagents for deactivating NAD-dependent ligase activity in the sample may be employed. Examples include:
  • These reagents may preferentially affect NAD-dependent ligase activity and not significantly affect, or affect to a lesser degree, ATP-dependent ligase activity in the sample.
  • These reagents may inhibit NAD-dependent ligase activity where the ligase is not already “charged” with NAD.
  • charge is meant that an AMP group derived from NAD is covalently attached to the NAD-dependent ligase, typically via a conserved lysine residue within the KXDG motif of the NAD-dependent ligase.
  • a dummy or false substrate may also be employed in the methods of the invention.
  • This false substrate can be acted upon by the ligase but is not detected and does not impair subsequent detection of the nucleic acid substrate molecule of the invention.
  • the false substrate does not include the primer and/or probe binding sites found in the nucleic acid substrate molecules of the invention.
  • the methods may incorporate both a chemical reagent to inhibit NAD-dependent ligase activity and a false substrate molecule.
  • nucleic acid substrate is after the NAD-dependent ligase has been inactivated or removed to prevent the NAD-dependent ligase from acting on the substrate.
  • the sample is then incubated under conditions suitable for nucleic acid modifying activity. This may involve incubation at an optimum temperature for nucleic acid modifying activity.
  • the sample may be incubated at a temperature between around 15 and 40 degrees Celsius, such as around 37 degrees Celsius. This may be for any suitable period of time, for example between 5 and 60 minutes, such as around 5, 10, 15, 20, 25 or 30 minutes.
  • the nucleic acid modifying activity may be inactivated prior to the modified nucleic acid molecule detection step. This may be achieved by elevating the temperature, for example to a temperature above 60 degrees Celsius, such as 95 degrees Celsius for a suitable time period. This may be a relatively short time period such as 1 , 2, 3, 4, 5, 10, 15 or more minutes.
  • Step d requires treating the sample, for example by adding UDG to the sample or heating the sample if a substrate is used which incorporates a heat-labile complementary strand or adding a nuclease if a protected first and second strand is utilised in order to render the complementary strand no longer a template strand.
  • the complementary strand of the nucleic acid substrate may comprise uracil bases such that upon treatment with uracil DNA glycosylase (UDG) enzyme the complementary strand is no longer a template strand. This ensures that, during the subsequent detection steps, typically a subsequent amplification reaction (a step in ETGA assays), the polymerase used in the amplification reaction is unable to amplify the complementary strand using the first strand as a primer.
  • UDG uracil DNA glycosylase
  • the method then requires performing a nucleic acid amplification step in order to amplify any ligated first and second strand in the sample (due to ligase activity in the sample, typically ATP-dependent ligase activity because the NAD-dependent ligase activity has already been removed or deactivated) and/or extended first strand in the sample (due to polymerase activity in the sample).
  • Amplification techniques are discussed in greater detail herein and include PCR, specifically quantitative PCR.
  • the final step of the method relies upon detecting and discriminating the amplification products to detect polymerase and/or ligase activity in a sample.
  • the presence of polymerase activity in the sample but no ligase activity indicates the presence of bacteria in the sample but not fungi. This is because the bacterial NAD-dependent ligase activity has already been removed/inactivated. If fungi were present in the sample, ATP- dependent ligase activity would be detected.
  • the presence of ligase activity in the sample indicates the presence of fungi.
  • Bacterial NAD-dependent ligase activity has already been removed/inactivated in the method and so the detection and/or quantification of ligated nucleic acid substrate indicates a fungal source of (ATP-dependent) ligase activity.
  • the presence of both polymerase and ligase activity in the sample indicates the presence of fungi.
  • bacterial NAD-dependent ligase activity has already been removed/inactivated in the method and so the detection and/or
  • quantification of ligated nucleic acid substrate indicates a fungal source of (ATP- dependent) ligase activity.
  • the presence of polymerase activity in addition to the ligase activity could be provided either by fungi or bacteria.
  • the relative levels of nucleic acid modifying activity will assist in discriminating the type of microorganism in the sample.
  • the inventors have observed that fungal ligase activity may be significantly faster than polymerase activity.
  • relative levels (of the extended and ligated products) may permit discrimination of fungi from bacteria.
  • a sample containing predominantly ligase activity but also limited polymerase activity is likely to contain only fungi, whereas a sample containing more similar levels of ligase and polymerase activity is likely to be a mixed sample containing both fungi and bacteria.
  • Relative levels can be determined with reference to levels of amplification products, for example by a simple comparison of cycle threshold (Ct) value.
  • step f of the method may comprise the addition of a first probe which hybridizes to a target sequence comprising the mismatched region contained within the second strand together with a second probe which hybridizes to a target sequence comprising the sequence corresponding to the mismatched region in the complementary strand.
  • Discriminating amplification products from ligase or polymerase activity relies upon detecting hybridization of the probes to their respective target sequences.
  • the probes may be differently labelled for example with detectably distinct fluorophores.
  • the probes may be incorporated into amplification primers in some embodiments.
  • the invention provides a method of detecting the absence or presence of a micro-organism in a sample comprising:
  • polymerase and/or ligase activity if present in the sample
  • sample in the context of the present invention is defined to include any sample in which it is desirable to test for the presence of a microorganism, such as a fungus (e.g. a yeast) or a bacterium, expressing nucleic acid modifying activity.
  • a microorganism such as a fungus (e.g. a yeast) or a bacterium, expressing nucleic acid modifying activity.
  • the sample may comprise, consist essentially of or consist of a clinical sample, such as a blood sample.
  • the methods of the invention are particularly applicable to the rapid determination of negative blood cultures.
  • the sample may comprise a blood culture sample from a patient suspected of suffering from, or being screened for, a bloodstream infection.
  • the sample may be any suitable volume such as 1 to 10ml, preferably a 1 ml blood culture sample.
  • the sample may be or comprise an in vitro assay system for example.
  • Samples may comprise, consist essentially of or consist of beverage or food samples or preparations thereof, or pharmaceutical or cosmetic products such as personal care products including shampoos, conditioners, and moisturisers etc., all of which are tested for microbial contamination as a matter of routine.
  • the sample may comprise, consist essentially of or consist of tissue or cells and may comprise, consist essentially of or consist of a sputum or a blood sample or a platelet sample for example.
  • the methods and kits of the invention may be used to monitor contamination of surfaces, such as for example in locations where food is being prepared.
  • Contamination is indicated by the presence of microbial nucleic acid modifying activity.
  • the contamination may be from any microbial source, in particular bacterial or fungal (e.g yeast) contamination.
  • the invention is also useful in monitoring environmental conditions such as water supplies, wastewater, marine environments etc.
  • the invention is also useful in monitoring bacterial growth in fermentation procedures and in air sampling where bacteria or spore content can be assessed in hospital, industrial facilities or in biodefence applications.
  • the sample contains a non-microorganism source of polymerase and/or ligase activity and the method comprises deactivating the non-micro-organism source of polymerase and/or ligase activity prior to adding the nucleic acid substrate to the sample.
  • the source may comprise mammalian cells, such as blood cells in some embodiments.
  • deactivating the non-microorganism source of polymerase and/or ligase activity prior to adding the nucleic acid substrate to the sample comprises treating the sample under high pH conditions in a manner such that micro-organism polymerase and/or ligase activity is not significantly affected.
  • the duration of the high pH conditions is typically less than 20 minutes and may be not more than 1 0, 9, 8, 7, 6 or 5 minutes and may be around 5, 6, 7, 8, 9 or 1 0 minutes.
  • the treatment is carried out for between around 2 and 1 5 minutes, such as around 5 minutes.
  • any suitable reagent may be added to the sample in order to provide high pH conditions.
  • the high pH conditions comprise contacting the sample with an alkali.
  • NaOH or Na2C03 is used.
  • the concentration of the NaOH or Na2C03 is around 5mM or greater.
  • the high pH conditions typically inhibit the activity of nucleic acid modifying enzymes including ATP-dependent ligase and polymerases from non-microorganism sources such as mammalian cells, but do not inhibit the activity of the microbial ligases or
  • High pH is generally a pH of at least around 10, such as around 10, 1 1 , 12, 13 or 14.
  • Low pH is generally a pH of less than or equal to around 4, such as around 4, 3, 2, or 1 .
  • around is meant 0.5 of a pH unit either side of the stated value. Altering the pH of the sample may be achieved using any suitable means, as would be readily appreciated by one skilled in the art.
  • Microbial enzymes such as polymerases and ligases may be resistant to extremes of pH, whereas mammalian ligases may be inactivated under the same pH conditions. This permits selective detection of microbial ligases in a sample containing both mammalian cells and microbial cells.
  • the conditions that inhibit the activity of non-microorganism nucleic acid modifying activity, such as ATP-dependent ligase, from mammalian cells but which do not inhibit the activity of the microorganism source of nucleic acid modifying activity, such as microbial ligases comprise treating the sample with sodium hydroxide (NaOH) or sodium carbonate (Na2C03).
  • Such agents can readily be used, as shown herein, to increase the pH of the sample to high pH thus inactivating mammalian ligase activity whilst leaving the microbial (fungal and bacterial) ligases active.
  • Suitable concentrations and volumes of the appropriate agent can be applied by a skilled person. In certain embodiments, however, the NaOH is at least around 5mM NaOH.
  • the pH is around 12 to inactivate mammalian nucleic acid modifying activity (such as polymerase and/or ATP-dependent ligase activity), but not microbial nucleic acid modifying activity (such as polymerase and/or ligase activity).
  • pH conditions may be increased to at least around 1 1 , or at least 1 1 .2. This treatment may result in lysis of micro-organisms in the sample and thus lead to nucleic acid modifying activity (e.g. polymerase and/or ligase) release into the sample. This permits detection of nucleic acid modifying activity (e.g.
  • polymerases and/or ligases in the sample, originating from the micro-organism, without the need for a separate cell lysis step.
  • mammalian ligases such as blood ATP-dependent ligases
  • the methods include a separate step for lysing microorganisms in the sample, as discussed in greater detail herein below.
  • the treatment under high pH conditions is stopped by adding a reagent to lower the pH.
  • Suitable reagents include a buffer and/or an acid.
  • the buffer comprises a Tris-HCI buffer (e.g. pH 7.2 or 8).
  • suitable agents for lowering the pH include acids such as hydrochloric acid (HCI) and sulphuric acid (H2S04). These (and other) acids may be incorporated into a buffer as would be readily appreciated by one skilled in the art.
  • nucleic acid amplification step may comprise, consist essentially of or consist of a nucleic acid amplification step. This serves to make the methods of the invention maximally sensitive.
  • amplification techniques are well known in the art, and include methods such as PCR (in particular qPCR), NASBA (Compton, 1991 ), 3SR (Fahy et al., 1991 ), Rolling circle replication, Transcription Mediated Amplification (TMA), strand displacement amplification (SDA) Clinical Chemistry 45: 777-784, 1999, the DNA oligomer self-assembly processes described in US6261846 (incorporated herein by reference), ligase chain reaction (LCR) (Barringer et al., 1990), selective amplification of target polynucleotide sequences (US 6410276), arbitrarily primed PCR (WO 90/06995), consensus sequence primed PCR (US 4437975), invader technology,
  • nick displacement technology and nick displacement amplification (WO 2004/067726).
  • the list above is not intended to be exhaustive. Any nucleic acid amplification technique may be used provided the appropriate nucleic acid product is specifically amplified. Similarly, sequencing based methodologies may be employed in some embodiments to include any of the range of next generation sequencing platforms. Amplification is achieved with the use of amplification primers specific for the sequence of the modified nucleic acid molecule which is to be detected. In order to provide specificity for the nucleic acid molecules primer binding sites corresponding to a suitable region of the sequence may be selected.
  • nucleic acid molecules may also include sequences other than primer binding sites which are required for detection of the novel nucleic acid molecule produced by the modifying activity in the sample, for example RNA Polymerase binding sites or promoter sequences may be required for isothermal amplification technologies, such as NASBA, 3SR and TMA.
  • One or more primer binding sites may bridge the boundary of the mismatch region of the substrate nucleic acid molecule such that an amplification product is only generated if one or other of ligation/extension has occurred, for example.
  • primers may bind either side of the mismatch region and direct amplification of this region with probe based detection of the nature of the mismatch.
  • Primers and the substrate nucleic acid molecule(s) may be designed to avoid non-specific amplification (e.g. of genomic DNA in the sample).
  • the primers may incorporate synthetic nucleotide analogues as appropriate or may be RNA or PNA based for example, or mixtures thereof.
  • the primers may be labelled, such as with fluorescent labels and/or FRET pairs, depending upon the mode of detection employed.
  • Probes may be utilised, again which may be labelled, as desired.
  • the methods of the invention are carried out using nucleic acid amplification techniques in order to detect the extended or ligated nucleic acid molecule produced as a direct result of the action of nucleic acid-modifying activity on the substrate nucleic acid molecule which indicates the presence of a microorganism in the sample.
  • the technique used is selected from PCR (including qPCR), NASBA, 3SR, TMA, SDA and DNA oligomer self-assembly.
  • Detection of the amplification products may be by routine methods, such as, for example, gel electrophoresis but in some embodiments is carried out using real-time or end-point detection methods. Preferably quantitative methods are employed.
  • a number of techniques for real-time or end-point detection of the products of an amplification reaction are known in the art. These include use of intercalating fluorescent dyes such as SYBR Green I (Sambrook and Russell, Molecular Cloning - A Laboratory Manual, Third edition), which allows the yield of amplified DNA to be estimated based upon the amount of fluorescence produced. Many of the real-time detection methods produce a fluorescent read-out that may be continuously monitored; specific examples including molecular beacons and fluorescent resonance energy transfer probes. Realtime and end-point techniques are advantageous because they keep the reaction in a "single tube". This means there is no need for downstream analysis in order to obtain results, leading to more rapidly obtained results.
  • Real-time and end-point quantitation of PCR reactions may be accomplished using the TaqMan® system (Applied Biosystems), see Holland et al; Detection of specific polymerase chain reaction product by utilising the 5'-3' exonuclease activity of Thermus aquaticus DNA polymerase; Proc. Natl. Acad. Sci. USA 88, 7276-7280 (1991 ), Gelmini et al. Quantitative polymerase chain reaction-based homogeneous assay with flurogenic probes to measure C-Erb-2 oncogene amplification. Clin. Chem. 43, 752-758 (1997) and Livak et al. Towards fully automated genome wide polymorphism screening. Nat. Genet.
  • This type of probe may be generically referred to as a hydrolytic probe. Suitable hydrolytic/Taqman probes for use in real time or end point detection are also provided. The probe may be suitably labelled, for example using the labels detailed herein.
  • Molecular Beacon system see Tyagi & Kramer. Molecular beacons - probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303-308 (1996) and Tyagi et al.
  • the beacons are hairpin-shaped probes with an internally quenched fluorophore whose fluorescence is restored when bound to its target. These probes may be referred to as hairpin probes.
  • a further real-time fluorescence based system which may be incorporated in the methods of the invention is the Scorpion system, see Detection of PCR products using self-probing amplicons and fluorescence by Whitcombe et al. Nature Biotechnology 17, 804 - 807 (01 Aug 1999).
  • Additional real-time or end-point detection techniques which are well known to those skilled in the art and which are commercially available include Lightcycler® technology, Amplifluour® primer technology, DzyNA primers (Todd et al., Clinical Chemistry 46:5, 625-630 (2000)), or the PlexorTM qPCR and qRT-PCR Systems.
  • the products of nucleic acid amplification are detected using real-time or end point techniques.
  • the real-time technique consists of using any one of hydrolytic probes (the Taqman® system), FRET probes (Lightcycler® system), hairpin primers (Amplifluour® system), hairpin probes (the Molecular beacons system), hairpin probes incorporated into a primer (the Scorpion® probe system), primers incorporating the complementary sequence of a DNAzyme and a cleavable fluorescent DNAzyme substrate (DzYNA), Plexor qPCR and oligonucleotide blocking systems.
  • Amplification products may be quantified to give an approximation of the microbial nucleic acid modifying activity in the sample and thus the level of microorganisms in the sample.
  • "absence or presence” is intended to encompass quantification of the levels of microorganisms in the sample.
  • the reaction mixture will contain all of; the sample under test, the substrate nucleic acid molecule(s), reagents, buffers and enzymes required for amplification of the modified nucleic acid molecule optionally in addition to the reagents required to allow real time or end-point detection of amplification products.
  • the entire detection method for the nucleic acid modifying activity may occur in a single reaction, with a quantitative output, and without the need for any intermediate washing steps.
  • Use of a "single tube” reaction is advantageous because there is no need for downstream analysis in order to obtain results, leading to more rapidly obtained results. Furthermore keeping the reaction in a "single tube” environment reduces the risk of cross
  • the methods of the invention may be carried out in step-wise fashion.
  • a first step it may first be necessary to prepare the sample in a form suitable for use in the method of the invention.
  • selective cell lysis or increasing cellular permeability may be required.
  • Capture of specific nucleic acid modifying activity, such as polymerase or ligase may also be desirable again as described herein.
  • Other (sources of) nucleic acid modifying activity, such as nuclease activity may be inhibited etc.
  • the methods of the invention are intended to assist in determining the absence or presence of viable microorganisms in a sample rapidly and economically.
  • the methods are much quicker than conventional blood culture testing and may benefit from an indication of when the blood culture can be sampled in order to perform the ETGA methods.
  • the invention further provides a blood culture bottle for micro- organism culture comprising :
  • the visual indicator indicates a pre-determined period of time at the end of which the sample is ready for further testing.
  • the visible indicator comprises a light source such as a light emitting diode (LED).
  • the visual indicator changes colour after the pre-determined period of time is reached. For example, the colour change may be from red to green.
  • the pre- determined period of time may be set according to requirements.
  • the period of time is set between (and including) around 4 to 1 6 hours, such as 6 to 1 2 hours, or 4, 5, 6, 7, 8, 9, 1 0, 1 1 or 12 hours.
  • around is meant plus or minus 30 minutes.
  • the methods of the invention may include culturing the sample in a blood culture bottle as described herein prior to performing the method. DESCRIPTION OF THE FIGURE
  • Figure 1 is a schematic representation of a "multi-detection" nucleic acid substrate permitting detection and identification of ligase and polymerase activity.
  • Figure 1 shows a nucleic acid substrate (1 ) of the invention.
  • the multi-detection substrate consist of three oligonucleotides; a first single strand (2), a second single strand downstream of the first strand (3) and a strand complementary to the first and second strands (4).
  • the first strand (2) is complementary to the 3' end region of the complementary strand (4).
  • the second strand (3) is also complementary to the complementary strand (4) apart from a short internal region, typically of 6-12 bases, that is not complementary ((5) and shown in black and boxed); the 5' and 3' ends of the second strand (3) therefore still base pair strongly with the complementary strand (4).
  • the second strand (3) also carries a 5' phosphate modification (allowing ligation).
  • the complementary strand (4) contains uracil bases that can be removed using UDG enzyme.
  • the first strand (2) When exposed to a mixture containing a ligase, the first strand (2) will be ligated to the second strand (3) creating a template containing the mismatched sequence; this is the ligated substrate.
  • the second strand (3) When exposed to a sample containing polymerase, the second strand (3) will be displaced (or proof-read and corrected) by the polymerase extending the DNA strand from the first strand (2) using the complementary strand (4) as the template; this is the polymerase extended substrate.
  • UDG enzyme When added to a PCR amplification reaction containing UDG enzyme and different fluorescent probes specific for the ligated substrate (eg. Cy3 labelled) and the polymerase extended substrate (e.g. FAM labelled), it will be possible to identify whether the template was generated by polymerase or ligase.
  • UDG enzyme must be used prior to the PCR amplification steps to prevent Taq polymerase from extending the DNA strand from the first strand (2) to create an amplifiable template

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Abstract

Cette invention concerne des procédés permettant de détecter l'absence ou la présence de micro-organismes dans un échantillon, lesdits procédés reposant sur des substrats d'acides nucléiques spécifiques. En particulier, les substrats d'acides nucléiques comprennent un premier brin, un second brin (en aval dans le sens 5' à 3' du premier brin) et un brin complémentaire qui est complémentaire des premier et second brins pour que les premier et second brins s'hybrident au brin complémentaire pour produire une structure sensiblement double brin et qui permet de déterminer une activité polymérase et/ou ligase et de les distinguer l'une de l'autre. Des kits associés et des flacons de culture de sang sont en outre décrits.
PCT/GB2015/052005 2014-07-10 2015-07-10 Détection de micro-organismes viables WO2016005767A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
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