Patent Application
20210024877
The present invention relates generally to the field of separating microorganisms from non-microorganism cells in a sample and methods 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 where the sample also contains non-microorganism sources of enzyme activity. The invention relies upon effective isolation of the microorganism source of enzymatic activity. 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 also relates to kits comprising reagents useful for carrying out the methods.
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.
Detection of adenylate kinase as an indicator of viability has also been proposed (Squirrel) D J, Murphy M J, Leslie R L, Green J C D: A comparison of ATP and adenylate kinase as bacterial cell markers: correlation with agar plate counts). 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.
In WO2009/007719, NAD-dependent ligases are described as a useful indicator of the presence of a 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.
WO2011/130584 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. Such assays have been termed “ETGA assays”, where ETGA stands for Enzymatic Template Generation and Amplification. A problem with ETGA assays for viable microorganisms in crude samples is the presence of contaminating polymerase activity outside the microorganisms arising from host (e.g. human) cells and dead microorganisms. The ETGA assay is unable to distinguish microorganism polymerase activity from that of the host or from dead microorganisms.
WO2010/119270 describes a method for removing DNA ligase activity outside intact microorganisms.
WO2011/070507 describes the selective lysis of animal cells using a non-ionic detergent and a buffer.
WO/2017/182775 describes a method of detecting the absence or presence of a microorganism in a sample that may also contain non-microorganism cells comprising the selective lysis of non-microorganism cells, filtering the lysate and detecting the absence or presence of microorganisms retained within or upon the filter.
The use of magnetic beads coated with specific binding moieties such as antibodies is also known. The specificity of these products is defined by the specificity of the antibody or other binding ligand, which is generally chosen for a particular purpose to be highly specific to allow the isolation of a particular microorganism.
WO03/102184 describes methods, compositions and kits for concentrating or separating cells (e.g. bacteria) using flocculating agents, such as polyamines or cationic detergents, to form complexes with cells causing them to aggregate. The separation of the aggregated cells can be effected with a solid phase which is capable of binding the cells, such as magnetic beads.
WO01/53525 describes a method of isolating cells (e.g. microorganisms) from a sample which method comprises binding the cells to a solid support by means of a carbohydrate ligand immobilised on the solid support. A kit for performing such a method is sold by DiaSorin Molecular (“Bugs′n Beads™” kit).
Other kits for isolating microorganisms include ApoH-Technologies Peps6 magnetic beads. The beads are coated with the synthetic molecule, Peps6, which is derived from the Apolipoprotein H protein (ApoH), also known as β-2 glycoprotein.
The present inventors have recognised that in samples taken from subjects suspected of carrying a microbial infection there are much greater levels of nucleated blood cells (leukocytes) than previously imagined even though the majority of samples are not in fact from infected subjects. This has led to the requirement for improved methods of separating potential microbes from blood cells, in particular leukocytes, in blood samples taken from patients screened for infection. The invention relates to the separation of microorganisms from non-microorganism cells in a sample by selectively capturing microorganisms with particles (e.g. magnetic particles) forming particle-microorganism complexes and separating the particle-microorganism complexes from the non-microorganism cells (e.g. using a magnetic field). This provides a way to address the issue described above enabling the detection of the absence or presence of microorganisms in the sample. The inventors have surprisingly found that this separation can be achieved with particles (e.g. magnetic particles) that are not coated with ligands. The inventors have discovered that certain reagents are particularly useful in the methods to ensure good separation of microorganisms from non-microorganism cells, in particular in complex samples such as blood, milk and urine.
The invention provides a method of separating microorganisms from non-microorganism cells in a non-microorganism cell-containing sample, the method comprising: a) incubating the sample with particles having an outer surface to form particle-microorganism complexes; and b) separating the particle-microorganism complexes from the non-microorganism cells.
The invention provides a method of separating microorganisms from non-microorganism cells in a non-microorganism cell-containing sample, the method comprising: a) incubating the sample with particles to form particle-microorganism complexes, wherein the particles have an outer polymeric surface; and b) separating the particle-microorganism complexes from the non-microorganism cells.
The invention provides a method of separating microorganisms from non-microorganism cells in a non-microorganism cell-containing sample, the method comprising: a) incubating the sample with particles to form particle-microorganism complexes, wherein the step of incubating is performed in the presence of sodium polyanethol sulfonate and/or a reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample; and b) separating the particle-microorganism complexes from the non-microorganism cells.
The invention provides a method of separating microorganisms from non-microorganism cells in a non-microorganism cell-containing sample, the method comprising: a) incubating the sample with particles to form particle-microorganism complexes, wherein the step of incubating is performed in the presence of sodium polyanethol sulfonate and/or a detergent;
and b) separating the particle-microorganism complexes from the non-microorganism cells.
The invention provides a method of separating microorganisms from non-microorganism cells in a non-microorganism cell-containing sample, the method comprising: a) incubating the sample with particles to form particle-microorganism complexes; and b) separating the particle-microorganism complexes from the non-microorganism cells; wherein the particles have an outer surface that is not coated with any of (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from Apolipoprotein H protein, (iv) a Mannose Binding Lectin protein.
In the methods, the step of incubating may be performed in the presence of sodium polyanethol sulfonate and/or a reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample.
In the methods, the step of incubating may be performed in the presence of sodium polyanethol sulfonate and/or a detergent. A detergent is an example of a reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample.
In the methods, the method may further comprise washing the separated particle-microorganism complexes to remove non-microorganism cells or lysate; optionally wherein the separated particle-microorganism complexes are washed with a solution comprising a detergent and/or sodium chloride.
The reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample may comprise a combination of a detergent and one or more enzymes. The one or more enzymes may comprise a proteinase and/or a DNAse. Suitable detergents and enzymes are discussed herein.
In the methods, step b) may be performed by any suitable means of separation. For example, separation may be achieved using a magnetic field to attract the particle-microorganism complexes or centrifugation.
In the methods, step b) may further comprise removing the non-microorganism cells from the particle-microorganism complexes.
In the methods, step a) may be preceded by selectively lysing non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample.
In the methods, selectively lysing non-microorganism cells in the sample whilst retaining intact any microorganisms present in the sample may comprise freezing and thawing the sample.
In the methods, selectively lysing non-microorganism cells in the sample whilst retaining intact any microorganisms present in the sample may comprise adding a detergent.
In the methods, step a) may be performed in the presence of a buffer. The buffer may have a pH between 7.4 and 8.5.
In the methods, step a) may be performed in the presence of sodium chloride. The sodium chloride may be present at a concentration of between 50 and 500 mM. Preferably, the sodium chloride may be present at a concentration around 150 mM.
In the methods, the reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample may be a detergent. In the methods, the detergent may be non-ionic. In the methods, the detergent may not be conjugated to the particles capable of forming complexes with microorganisms. Thus, typically the detergent forms part of a solution to which the particles are added and does not form part of the particles themselves.
In the methods, the particles may have a diameter of between 0.1 and 3 μm or between 0.1 and 2 μm. Preferably, the particles have a diameter of between 0.1 and 1.0 μm
In the methods, the particles may be (and typically are) magnetic. The particles may be superparamagnetic. The particles may comprise iron oxide. The iron oxide may comprise magnetite and/or maghemite. The iron oxide may not comprise a 1:1, 2:1, 3:1 or 4:1 ratio of Fe2+ and Fe3+.
The outer surface of the particles capable of forming complexes with microorganisms may comprise a polymer; optionally the polymer may be carbon-based. The polymer may not comprise an inorganic polymer. The polymer may comprise polystyrene and/or poly(styrene/divinyl benzene).
In the methods, the outer surface of the particles capable of forming complexes with microorganisms may comprise or be coated with any one or more of: i) carboxylic acid groups; ii) amino groups; iii) hydrophobic groups; and iv) streptavidin; optionally the carboxylic acid groups; ii) amino groups; iii) hydrophobic groups may not be part of a polypeptide.
In the methods, the microorganism may be a pathogenic microorganism. For example, the pathogenic microorganism may be a pathogenic bacterium or fungus.
In the methods, the non-microorganism cells may comprise red blood cells and/or white blood cells.
In the methods, the sample may comprise non-microorganism cells at a concentration of between 20,000 and 5 million cells per millilitre. The sample may comprise non-microorganism cells at a concentration of at least around 100,000 cells per millilitre.
Preferably, the sample may comprise non-microorganism cells at concentration of at least around 20,000 cells per millilitre.
The sample is one which contains, or is suspected to contain, microorganisms. The sample contains non-microorganism cells which can provide unwanted background when aiming to detect whether and potentially also identify and/or quantify microorganisms present in the sample. Thus, in some embodiments, the sample may comprise blood, urine, saliva or milk. The blood sample may be any sample containing blood cells. The blood sample may be whole blood or may comprise whole blood (e.g. blood broth).
The invention provides a method of separating microorganisms from non-microorganism cells in a non-microorganism cell-containing sample, the method comprising: (a) incubating the sample with particles (e.g. magnetic particles) to form particle-microorganism complexes; and (b) separating the particle-microorganism complexes from the non-microorganism cells (e.g. using a magnetic field).
In the methods, the particles (e.g. magnetic particles) may have an outer polymeric surface that is not coated with any of (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from Apolipoprotein H protein, (iv) Mannose Binding Lectin, (v) a polyamine or (vi) a cationic detergent.
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with any of (i) an antibody, (ii) a carbohydrate or (iii) an innate immune system protein.
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with any of (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from Apolipoprotein H protein, (iv) Mannose Binding Lectin, or (v) a flocculating agent (e.g. a flocculating agent as defined in WO 03/102184).
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with any of (i) an antibody, (ii) a carbohydrate, (iii) an innate immune system protein or (iv) a flocculating agent (e.g. a flocculating agent as defined in WO 03/102184).
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with a ligand.
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is coated with streptavidin and is not coated with a ligand.
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. an outer polymeric surface) that is coated only with streptavidin.
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. an outer polymeric) surface that is coated only with carboxyl groups.
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with any molecule or moiety capable of binding to a microorganism.
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. an outer polymeric surface) that is not coated with any molecules or moieties.
By “coated” is meant attached to the outer surface (e.g. outer polymeric surface). The skilled person would be aware of means for the attachment of molecules and chemical groups to the outer surface (e.g. outer polymeric surface) of the particles (e.g. magnetic particles).
The separation methods of the invention are useful for enabling detection of whether or not a microorganism is found in a sample that also contains non-microorganism cells. Once the microorganisms have been separated from potential sources of background signal they can then be specifically and sensitively detected using a range of techniques. Accordingly, the invention also provides a method of detecting the absence or presence of a microorganism in a sample that may also contain non-microorganism cells comprising: a) incubating the sample with particles to form particle-microorganism complexes; b) separating the particle-microorganism complexes from the non-microorganism cells; and c) detecting the absence or presence of microorganisms in the particle-microorganism complexes.
The invention also provides a method of detecting the absence or presence of a microorganism in a sample that may also contain non-microorganism cells comprising: a) incubating the sample with particles to form particle-microorganism complexes, wherein the particles have an outer polymeric surface; b) separating the particle-microorganism complexes from the non-microorganism cells; and c) detecting the absence or presence of microorganisms in the particle-microorganism complexes.
Relatedly the invention provides a method of detecting the absence or presence of a microorganism in a sample that may also contain non-microorganism cells comprising: a) incubating the sample with particles to form particle-microorganism complexes, wherein the step of incubating is performed in the presence of sodium polyanethol sulfonate and/or a reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample; b) separating the particle-microorganism complexes from the non-microorganism cells; and c) detecting the absence or presence of microorganisms in the particle-microorganism complexes.
Similarly, the invention provides a method of detecting the absence or presence of a microorganism in a sample that may also contain non-microorganism cells comprising: a) incubating the sample with particles to form particle-microorganism complexes, wherein the step of incubating is performed in the presence of sodium polyanethol sulfonate and/or a detergent; b) separating the particle-microorganism complexes from the non-microorganism cells; and c) detecting the absence or presence of microorganisms in the particle-microorganism complexes.
The invention further provides a method of detecting the absence or presence of a microorganism in a sample that may also contain non-microorganism cells comprising: a) incubating the sample with particles to form particle-microorganism complexes; b) separating the particle-microorganism complexes from the non-microorganism cells; and c) detecting the absence or presence of microorganisms in the particle-microorganism complexes; wherein the particles have an outer surface that is not coated with any of (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from Apolipoprotein H protein, (iv) a Mannose Binding Lectin protein.
In the methods, the step of incubating may be performed in the presence of sodium polyanethol sulfonate and/or a reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample.
In the methods, step c) may comprise (i) detecting an enzymatic activity of a nucleic acid molecule associated with the microorganism, (ii) detecting the microorganism directly by cytometry or microscopy, (iii) detecting the microorganism following cell culture, (iv) detecting the microorganism by PCR or (v) detecting the microorganism by nucleic acid sequencing.
In the methods, step c) may comprise the steps of: i) lysing the microorganisms in the particle-microorganism complexes; ii) incubating the lysate with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the microorganisms; and iii) specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying enzyme on the substrate nucleic acid molecule to indicate the absence or presence of the microorganism. In the methods, step (i) may comprise adding a lysis reagent containing the substrate nucleic acid molecule. In the methods, the nucleic acid modifying enzyme may comprise a DNA or RNA polymerase, optionally wherein the DNA polymerase is DNA polymerase I.
Since microorganisms are a common source of infection in a subject, the methods of the invention are useful for identifying infection caused by a microorganism. Accordingly, the invention also provides a method of detecting the absence or presence of a microorganism infection in a subject comprising performing any of the methods described herein (that detect microorganisms in a sample) on a sample from the subject.
The method may further comprise washing the separated particle-microorganism complexes to remove non-microorganism cells or lysate.
In the methods, step (b) may further comprise removing the non-microorganism cells from the particle-microorganism complexes.
In the methods, step b) may be performed by any suitable means of separation. For example, separation may be achieved using a magnetic field to attract the particle-microorganism complexes or centrifugation.
In the methods, step b) may further comprise removing the non-microorganism cells from the particle-microorganism complexes.
In the methods, step a) may be preceded by selectively lysing non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample.
In the methods, selectively lysing non-microorganism cells in the sample whilst retaining intact any microorganisms present in the sample may comprise freezing and thawing the sample.
In the methods, selectively lysing non-microorganism cells in the sample whilst retaining intact any microorganisms present in the sample may comprise adding a detergent.
In the methods, step a) may be performed in the presence of a buffer. The buffer may have a pH between 7.4 and 8.5.
In the methods, step a) may be performed in the presence of sodium chloride. The sodium chloride may be present at a concentration of between 50 and 500 mM. Preferably, the sodium chloride may be present at a concentration around 150 mM.
In the methods, the reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample may be a detergent. In the methods, the detergent may be non-ionic. In the methods, the detergent may not be conjugated to the particles capable of forming complexes with microorganisms. Thus, typically the detergent forms part of a solution to which the particles are added and does not form part of the particles themselves.
In the methods, the particles may have a diameter of between 0.1 and 3 μm or between 0.1 and 2 μm. Preferably, the particles have a diameter of between 0.1 and 1.0 μm
In the methods, the particles may be (and typically are) magnetic. The particles may be superparamagnetic. The particles may comprise iron oxide. The iron oxide may comprise magnetite and/or maghemite. The iron oxide may not comprise a 1:1, 2:1, 3:1 or 4:1 ratio of Fe2+ and Fe3+.
The outer surface of the particles capable of forming complexes with microorganisms may comprise a polymer; optionally the polymer may be carbon-based. The polymer may not comprise an inorganic polymer. The polymer may comprise polystyrene and/or poly(styrene/divinyl benzene).
In the methods, the outer surface of the particles capable of forming complexes with microorganisms may comprise or be coated with any one or more of: i) carboxylic acid groups; ii) amino groups; iii) hydrophobic groups; and iv) streptavidin; optionally the carboxylic acid groups; ii) amino groups; iii) hydrophobic groups may not be part of a polypeptide.
In the methods, the microorganism may be a pathogenic microorganism. For example, the pathogenic microorganism may be a pathogenic bacterium or fungus.
In the methods, the non-microorganism cells may comprise red blood cells and/or white blood cells.
In the methods, the sample may comprise non-microorganism cells at a concentration of between 20,000 and 5 million cells per millilitre. The sample may comprise non-microorganism cells at a concentration of at least around 100,000 cells per millilitre.
Preferably, the sample may comprise non-microorganism cells at concentration of at least around 20,000 cells per millilitre.
The sample is one which contains, or is suspected to contain, microorganisms. The sample contains non-microorganism cells which can provide unwanted background when aiming to detect whether and potentially also identify and/or quantify microorganisms present in the sample. Thus, in some embodiments, the sample may comprise blood, urine, saliva or milk.
The blood sample may be any sample containing blood cells. The blood sample may be whole blood or may comprise whole blood (e.g. blood broth).
The invention provides a method of detecting the absence or presence of a microorganism in a sample that may also contain non-microorganism cells comprising: (a) incubating the sample with particles (e.g. magnetic particles) to form particle-microorganism complexes; (b) separating the particle-microorganism complexes from the non-microorganism cells (e.g. using a magnetic field); and (c) detecting the absence or presence of microorganisms in the particle-microorganism complexes.
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with any of (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from Apolipoprotein H protein, (iv) a Mannose Binding Lectin protein, (v) a polyamine or (vi) a cationic detergent.
In the methods, the particles (e.g. magnetic particles) may have an outer polymeric surface that is not coated with any of (i) an antibody, (ii) a carbohydrate or (iii) an innate immune system protein.
In the methods, the particles (e.g. magnetic particles) may have an outer polymeric surface that is not coated with any of (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from Apolipoprotein H protein, (iv) Mannose Binding Lectin, or (v) a flocculating agent (e.g. a flocculating agent as defined in WO 03/102184).
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with a ligand.
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with any of (i) an antibody, (ii) a carbohydrate, (iii) an innate immune system protein or (iv) a flocculating agent (e.g. a flocculating agent as defined in WO 03/102184).
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is coated with streptavidin and is not coated with a ligand.
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is coated only with streptavidin.
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is coated only with carboxyl groups.
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with any molecule or moiety capable of binding to a microorganism.
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with any molecules or moieties.
In the methods, step (c) may comprise (i) detecting an enzymatic activity of a nucleic acid molecule associated with the microorganism, (ii) detecting the microorganism directly by cytometry or microscopy, or (iii) detecting the microorganism following cell culture.
The detection of the absence or presence of microorganisms in the particle-microorganism complexes according to all relevant aspects of the invention can be performed according to any desired method. The method may involve detecting the simple absence or presence of the one or more microorganisms. It may involve quantification of the microorganisms, if present. It may also involve characterisation of the nature of the microorganism in some embodiments. Thus, detection of bacteria and/or fungi may be performed. Discrimination of gram positive versus gram negative bacteria may also be performed. Identification and antimicrobial susceptibility of the organisms may also be performed.
Detection may occur after the removal (or recovery) of the microorganisms from the particle-microorganism complexes. Recovered microorganisms may be lysed prior to detection. Recovery may be of the intact microorganisms or of a lysate following lysis of the microorganisms (as discussed in further detail herein).
Preferably, detection occurs without prior removal (or recovery) of the microorganisms from the particle-microorganism complexes. This embodiment is particularly useful in applying the invention to magnetic bead-processing instrumentation.
The detection of the absence or presence of microorganisms may comprise detecting an enzymatic activity or a nucleic acid molecule associated with the microorganism; detecting the microorganism directly by cytometry or microscopy; or detecting the microorganism following cell culture.
Detection of nucleic acid molecules associated with microorganisms is known in the art and may be performed at the DNA or RNA level. It can be performed by any suitable method, such as amplification (e.g. PCR) or sequencing (in particular next generation sequencing). Such methods may take advantage of sequence divergence between microorganisms and non-microorganisms, such as human, DNA and RNA. Such methods may involve lysing the microorganisms (e.g. present in the form of particle-microorganism complexes) in order to release the nucleic acid component.
Direct detection of microorganisms is also known. This may involve cytometric analysis, for example by flow cytometry. It may involve use of microscopy, for example to visualise the microorganisms recovered from particle-microorganism complexes or to visualise microorganisms in particle-microorganism complexes.
Microorganism detection may also be performed following cell culture, in order to expand the number of microorganisms. Thus, the microorganisms initially captured within particle-microorganism complexes can be cultured for a set period of time, prior to detection. Culture methods may permit direct detection of microorganisms in the original sample.
However, in preferred embodiments, the detection of the absence or presence of microorganisms may comprise detecting an enzymatic activity associated with the microorganism. Suitable enzymatic activities are typically nucleic acid modifying activities and are discussed in greater detail herein.
Accordingly, in the methods, step (c) may comprise the steps of: (i) lysing the microorganisms in the particle-microorganism complexes; (ii) incubating the lysate with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the microorganisms; and (iii) specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying enzyme on the substrate nucleic acid molecule to indicate the absence or presence of the microorganism.
In the methods, step (i) may comprise adding a lysis reagent containing the substrate nucleic acid molecule.
Incubating the sample refers to contacting the sample with the particles under conditions conducive to the formation of particle-microorganism complexes. In some embodiments, the step of incubating the sample with particles (e.g. magnetic particles) comprises contacting the particles (e.g. magnetic particles) with the sample for a fixed period of time (e.g. 30 minutes) at a specified temperature (e.g. 37° C.) The incubation may be performed with or without shaking (e.g. by a platform shaker, orbital shaker or shaking incubator set at 500-1000 rpm).
Lysis of microorganisms in the particle-microorganism complexes permits detection of nucleic acid molecules or enzymes within the microorganisms, such as nucleic acid modifying enzymes. Lysis may be achieved by addition of a lysis mixture. The lysis mixture is generally useful in the methods of the invention. The lysis mixture may include a specific mixture of components to ensure efficient lysis of microorganisms without adversely affecting nucleic acid molecules and/or enzyme activity, such as nucleic acid modifying activity, within the cells. The components may be selected from carrier/serum proteins such as BSA, surfactants/detergents, metal halide salts, buffers, chelators etc. In its basic form, the lysis mixture of the invention may include the following components:
A suitable lysis mixture is set forth below:
L1: 252 mL in 360 mL LM
1.46% (w/v) BSA
0.15% Triton X100
0.15% Tween 20
L2: 36 mL in 360 mL LM
100 mM Ammonium sulphate
20 mM Magnesium sulphate heptahydrate
100 mM Potassium chloride
200 mM Tris-HCl [pH 8.0]
L3: 36 mL in 360 mL LM
0.1 μM PTO-AS oligo
0.1 μM PTO-S1 oligo
20 mM Tris-HCl [pH 8.5]
10 mM Potassium chloride
10 μM EDTA
10 mM dNTPs: 3.6 mL in 360 mL LM
PTO-IPC stock: ˜180 μL in 360 mL LM
H2O: ˜32.4 mL in 360 mL LM
By “PTO-AS oligo” is meant an antisense oligonucleotide comprising phosphorothioate nucleotides. By “PTO-S1 oligo” is meant a sense oligonucleotide comprising phosphorothioate nucleotides. The two oligonucleotides hybridise to one another to form the substrate nucleic acid molecule.
By “PTO-IPC” is meant an IPC molecule comprising phosphorothioate nucleotides.
Suitable substrate and IPC molecules are discussed in further detail herein.
Exemplary amounts and concentrations of each component are listed but may be modified as would be readily appreciated by one skilled in the art.
Lysis may also require disruption of the cells. For example, the cells may be disrupted using the lysis mixture in combination with physical and/or enzymatic means. Typically, however, the methods in which the cells are lysed avoid use of physical disruption. In some embodiments, physical disruption employs a disruptor. The disruptor may incorporate beads such as glass beads to lyse the cells. Suitable apparatus are commercially available and include the Disruptor Genie manufactured by Scientific Industries, Inc. Sonication may be utilised, for example applying an ultra sonic horn. Enzymatic disruption may require use of one or more agents selected from lysostaphin, lysozyme and/or lyticase in some embodiments.
Once the microorganisms, if present in the sample, are lysed, the released nucleic acid and/or enzymes may be detected to indicate whether microorganisms are present in the sample. In some embodiments, the lysate is incubated with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity (of the microorganisms). The absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying enzyme on the substrate nucleic acid molecule is then determined to indicate the absence or presence of the microorganism. The nucleic acid substrate molecule is designed according to the nucleic acid modifying activity that is to be detected. One skilled in the art is well able to design suitable substrate nucleic acid molecules. Although the initial sample contains non-microorganism sources of nucleic acid modifying activity, the methods of the invention prevent this contaminating activity acting on the substrate nucleic acid molecules.
The nucleic acid modifying enzyme may comprise a DNA or RNA polymerase, optionally wherein the DNA polymerase is DNA polymerase I.
The nucleic acid modifying enzyme may comprise a ligase, optionally wherein the nucleic acid modifying enzyme is an NAD-dependent ligase.
The invention further provides a method of detecting the absence or presence of a microorganism infection in a subject comprising performing the method of any of the methods described herein on a sample from the subject, optionally wherein the sample comprises blood from the subject.
The method may further comprise washing the separated particle-microorganism complexes to remove non-microorganism cells or lysate. The step of washing may remove inhibitors of the subsequent analysis e.g. PCR inhibitors. The step of washing may be performed under conditions that do not dissociate the particle-microorganism complexes.
According to the methods of the invention typical nucleic acid modifying activity that may be detected comprises polymerase and/or ligase activity. In certain embodiments, nucleic acid modifying enzyme comprises DNA or RNA polymerase. In some embodiments, the DNA polymerase comprises or is DNA polymerase I. In some embodiments, the nucleic acid modifying enzyme comprises a ligase. In certain embodiments, the nucleic acid modifying enzyme comprises or is an NAD-dependent or ATP-dependent ligase. NAD-dependent ligases are only found in (eu)bacteria and thus, detecting such activity may provide an additional level of specificity. This is discussed further in WO2009/007719 and WO2010/119270 (the pertinent disclosures of which are hereby incorporated). Other nucleic acid modifying activities relevant to viability may alternatively be measured such as phosphatase, kinase and/or nuclease activity.
In some embodiments, the action of the nucleic acid modifying activity on the substrate nucleic acid molecule produces an extended nucleic acid molecule. This may be by strand extension (polymerase activity) and/or by ligation of two nucleic acid molecules (ligase activity). In some embodiments, a substrate that can be acted upon by either polymerase or ligase is utilised since either activity is indicative of the presence of a microorganism in the sample. In some embodiments, the relevant activity can be distinguished in terms of the novel nucleic acid molecule that is produced.
The substrate may be a template for the nucleic acid modifying activity of the microorganisms. For example, the substrate may be a template for a DNA or RNA polymerase, optionally wherein the DNA polymerase is DNA polymerase I.
Suitable nucleic acid molecules which acts as a substrate for nucleic acid modifying activity of the microorganisms are described in WO2011/130584, WO2010/119270 and WO2009/007719 (the pertinent disclosures of which are hereby incorporated). In the case of phosphatase activity, suitable nucleic acid molecules are disclosed in WO2006/123154, which disclosure is hereby incorporated by reference.
In specific embodiments, the (substrate) nucleic acid molecule used in the methods of the invention is at least partially double stranded and comprises uracil residues in the complementary strand and the step of specifically determining the absence or presence of the modified nucleic acid molecule comprises adding Uracil DNA Glycosylase (UDG) to the sample in order to degrade the uracil residues in the complementary strand.
In certain embodiments, the (substrate) nucleic acid molecule comprises DNA. In certain embodiments, the (substrate) nucleic acid molecule comprises DNA and is partially double-stranded.
In some embodiments, the (substrate) nucleic acid molecule comprises a nucleic acid consisting of a sense oligonucleotide (DNA) strand and an antisense oligonucleotide (DNA) strand, wherein the two strands overlap to form a double stranded region and a single stranded portion of the antisense oligonucleotide strand acts as a template with the sense oligonucleotide strand of the double stranded region acting as a primer to create an extension product in the presence of polymerase activity;
In certain embodiments, the first strand of the partially double stranded (substrate) nucleic acid molecule comprises (or consists of) synthetic nucleotides (e.g. phosphorothioate nucleotides) and the second (complementary) strand comprises (or consists of) uracil residues and, optionally, synthetic nucleotides (e.g. phosphorothioate nucleotides). Preferably, the double stranded region encompasses the 3′ end regions of the first and second (complementary) strands. Preferably, the second (complementary) strand comprises a base (e.g. dideoxyCytidine) at its 3′ end that blocks DNA polymerase-mediated extension of the second strand. Such partially double stranded (substrate) nucleic acid molecules are described, for example, in Zweitzig et al., 2012 (Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes. Nucleic Acids Research 40, 14, e109, 1-12). Preferably, the double stranded region is at least 5, at least 10, at least 15, at least 20 or at least 25 nucleotides; optionally, the double stranded region is no more than 50 nucleotides. The first strand may be extended during an incubation step, as described herein, using unprotected (or standard) dNTPs by the polymerase activity of a microorganism in the sample to form an extended first strand that comprises unprotected (or standard) nucleotides. This step relies upon using the second strand as template (upstream of the region of complementarity between the first and second strands). Following the incubation step, the second (complementary) strand may be degraded by adding Uracil DNA Glycosylase (UDG) to the sample leaving the extended first strand as a single stranded molecule comprising synthetic nucleotides and unprotected nucleotides. Following degradation of the second strand, the extended first strand of the (substrate) nucleic acid molecule may be detected in an amplification step. The inventors have found that the use of a partially double stranded (substrate) nucleic acid molecule as described above improves the detection of a microorganism in the sample.
In some embodiments, the substrate nucleic acid molecule is pre-modified so as to protect it from nuclease activity i.e. the nucleic acid molecule is modified so as to protect it from nuclease activity before it is added to the assay. The inventors have determined that protection of the substrate nucleic acid molecule from nuclease activity is advantageous in the context of the assays of the invention. More specifically, incorporation of protected nucleic acid molecules into the methods of the invention improves sensitivity of detection. 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. In specific embodiments, the synthetic nucleotides comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides. Preferably, the synthetic nucleotides are phosphorothioate nucleotides. In certain embodiments, the synthetic nucleotides replace at least one up to all of the nucleotides in the nucleic acid molecule.
The (substrate) nucleic acid molecules may 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 a (novel detectable) nucleic acid molecule. The substrate may be extended and/or ligated in specific embodiments. Combinations of nucleic acid substrate molecules may be employed to permit detection of polymerase and ligase activity in some embodiments.
The nucleic acid substrate may be present in excess, and in particular in large molar excess, over the nucleic acid modifying activity (provided by the microorganisms) in the sample. Because a novel extended 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 inventors have previously investigated the use of an internal positive control (IPC) molecule in the context of their methods. Thus, according to all aspects, the invention may rely upon inclusion of an IPC molecule. In some embodiments, the IPC is included with the substrate nucleic acid molecule so that the IPC is exposed to identical conditions. In some embodiments, the IPC molecule is pre-modified so as to protect it from nuclease activity i.e. the nucleic acid molecule is modified so as to protect it from nuclease activity before it is added to the assay. The inventors have determined that protection of the IPC molecule from nuclease activity is advantageous in the context of the assays of the invention. 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. In specific embodiments, the synthetic nucleotides comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides. Preferably, the synthetic nucleotides are phosphorothioate nucleotides. In certain embodiments, the synthetic nucleotides replace at least one up to all of the nucleotides in the IPC molecule. Preferably, the substrate and IPC molecules are modified in the same manner as it is advantageous for them to behave similarly in the assays of the invention.
In some embodiments, the internal positive control (IPC) nucleic acid molecule comprises identical primer binding sites to the substrate nucleic acid molecule such that there is competition for primer binding in a nucleic acid amplification reaction containing both the nucleic acid molecule and the IPC.
In all methods of the invention specifically determining the absence or presence of the modified nucleic acid molecule may comprise, consist essentially of or consist of a nucleic acid amplification step. This serves to make the methods of the invention maximally sensitive. Such amplification techniques are well known in the art, and include methods such as PCR, 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 U.S. Pat. No. 6,261,846 (incorporated herein by reference), ligase chain reaction (LCR) (Barringer et al., 1990), selective amplification of target polynucleotide sequences (U.S. Pat. No. 6,410,276), arbitrarily primed PCR (WO 90/06995), consensus sequence primed PCR (U.S. Pat. No. 4,437,975), invader technology, strand 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, such as sequencing by synthesis of clonally amplified sequences (Illumine), pyrosequencing, 454 sequencing (Roche), nanopore sequencing (e.g. Oxford Nanopore), ion torrent (ThermoFisher) and single molecule real-time (SMRT) sequencing (Pacific Biosystems). The fact that a novel nucleic acid molecule is generated means that a sequencing approach can confirm the presence or otherwise of the modified nucleic acid molecule and also provide quantification of that molecule.
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. The skilled reader will appreciate that the 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 ligation/extension boundary of the substrate nucleic acid molecule such that an amplification product is only generated if ligation/extension has occurred, for example. Alternatively, primers may bind either side of the ligation/extension boundary and direct amplification across the boundary such that an amplification product is only generated (exponentially) if the ligated/extended nucleic acid molecule is formed. Primers and the substrate nucleic acid molecule(s) may be designed to avoid non-specific amplification (e.g. of genomic DNA in the sample).
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 detection method may require use of nucleotide probes in addition to primers, or as an alternative to primers. For example, a branched DNA assay, which does not require use of primers, may be employed in some embodiments.
In certain aspects, the methods of the invention are carried out using nucleic acid amplification techniques in order to detect the modified 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 micro-organism in the sample. In certain embodiments the technique used is selected from PCR, 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.
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. Real-time 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. Furthermore keeping the reaction in a “single tube” environment reduces the risk of cross contamination and allows a quantitative output from the methods of the invention. This may be particularly important in the context of the present invention where health and safety concerns may be of paramount importance (such as in detecting potential microbial infection in a patient samples for example).
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. 9, 341-342 (19995) (incorporated herein by reference). 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 below.
In the Molecular Beacon system, see Tyagi & Kramer. Molecular beacons—probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303-308 (1996) and Tyagi et al. Multicolor molecular beacons for allele discrimination. Nat. Biotechnol. 16, 49-53 (1998) (incorporated herein by reference), 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 (1 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 Plexor™ qPCR and qRT-PCR Systems.
Thus, in further aspects of the invention the products of nucleic acid amplification are detected using real-time or end point techniques. In specific embodiments of the invention 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. Thus, “absence or presence” is intended to encompass quantification of the levels of microorganisms in the sample.
The inventors have further discovered that the optimal temperature for measuring nucleic acid modifying activity of the microorganisms may not be the same as the optimal temperature for lysis of microorganisms. Thus, in some embodiments, lysis of the microorganisms is performed at a lower temperature than the step of incubating the lysate with a nucleic acid molecule that acts as a substrate for nucleic acid modifying activity of the microorganisms. As already discussed, in some embodiments of the invention, the substrate nucleic acid molecule is included in the lysis reagent used to lyse the microorganisms. Such embodiments are consistent with the differing temperature preferences. Thus, even though the substrate nucleic acid molecule may be included in the lysis reagent, the initial lower temperature does not adversely affect the subsequent incubation at higher temperature, at which the substrate is modified by the nucleic acid modifying activity released from the microorganisms. Accordingly, in some embodiments the method involves a step of lysis of the microorganisms in which the lysis reagent contains a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the microorganisms. This step is performed at a lower temperature than the subsequent step of incubating the lysate with the substrate nucleic acid molecule to enable the activity of the enzymes released from the microorganisms. Thus, the substrate is exposed to the initial lower temperature, followed by a higher temperature under which enzyme activity is enhanced.
In some embodiments, the step of incubating the lysate with a nucleic acid molecule that acts as a substrate for nucleic acid modifying activity of the microorganisms is performed at a temperature of at least around 30° C. The temperature may be optionally between around 30° C. and 40° C. or between around 32° C. and 37° C., such as around 37° C.
In additional or alternative embodiments, the step of lysis of the microorganisms is performed at a temperature of no more than around 30° C., optionally between around 15° C. and 30° C. or between around 18° C. and 25° C., such as around 18, 19, 20, 21, 22, 23, 24 or 25° C. In some embodiments, all steps prior to incubating the lysate with a nucleic acid molecule that acts as a substrate for nucleic acid modifying activity of the microorganisms are performed at a temperature of no more than around 30° C. The temperature may optionally be between around 15° C. and 30° C. or between around 18° C. and 25° C., such as around 18, 19, 20, 21, 22, 23, 24 or 25° C.
Such a method may incorporate any one or more up to all of the embodiments described in relation to the various aspects of the invention.
In some embodiments, the method is further characterised in that the step of incubating the lysate with a substrate nucleic acid molecule is performed at a temperature of at least around 30° C., optionally between around 30° C. and 40° C. or between around 32° C. and 37° C., such as around 37° C.
In additional or alternative embodiments, each of steps prior to incubating the lysate with a substrate nucleic acid molecule is performed at a temperature of no more than around 30° C., optionally between around 15° C. and 30° C. or between around 18° C. and 25° C., such as around 18, 19, 20, 21, 22, 23, 24 or 25° C.
Prior to the step of incubating the sample with magnetic particles to form particle-microorganism complexes (i.e. prior to step (a)), the method may comprise selectively lysing non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample.
The step of selectively lysing non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample may comprise adding a combination of a detergent and one or more enzymes to the sample. The one or more enzymes may comprise a proteinase and/or a DNAse, optionally wherein the proteinase is proteinase K.
The step of selective lysis of non-microorganism cells in the sample whilst retaining intact any microorganisms present in the sample may prevent enzymatic activity from non-microorganism cells, such as leukocytes, falsely indicating the presence of microorganisms in the sample. Such selective lysis can be achieved by any suitable means as discussed further herein. Any suitable reagent that lyses non-microorganisms, in particular mammalian cells, present in the sample but does not lyse microorganisms in the sample may be utilised. The reagent may include a surfactant or detergent in some embodiments, such as a non-ionic detergent. Suitable examples include polyethylene glycol sorbitan monolaurate (Tween 20), for example at 5% w/v. The reagent may include a saponin, for example at 5% w/v. The reagent may include a metal halide salt, such as sodium chloride, for example at 8.5 g/l. The reagent may include a mixture of all three components. The sample may be mixed with the reagent under suitable conditions to ensure lysis of non-microorganism cells, in particular mammalian cells, if present in the sample but no (or insignificant) lysis of microorganisms if present in the sample. The sample may be exposed to the reagent for a period of between around 5 and 30 minutes, such as 5, 10, 15, 20, 25 or 30 minutes. This step may be performed at any suitable temperature, for example between 15 and 30 degrees Celsius or at room temperature.
In some embodiments, according to all aspects of the invention, selective lysis of non-microorganism cells in the sample whilst retaining intact any microorganisms present in the sample comprises adding a combination of a detergent and one or more enzymes to the sample. Without wishing to be bound by any particular theory, the detergent selectively permeabilises non-microorganism cell membranes, whereas the microorganisms are protected by virtue of their cell wall. The enzymes are useful for breaking down released intracellular material and other cellular debris and may contribute to preventing carry over of released enzymatic activity. In some embodiments, the one or more enzymes comprise a proteinase and/or a nuclease. Suitable proteinases include proteinase K. Suitable nucleases include DNAses. In one embodiment, the reagent used to selectively lyse non-microorganism cells comprises a combination of triton X-100 and proteinase K. More specifically the lysis reagent may comprise 0.25% Triton X-100 and 4.8 μg/mL Proteinase K.
It is important to inactivate any relevant enzymatic activity released if the non-microorganism cells are lysed. The inventors have devised methods in which high pH conditions are utilised to ensure effective inactivation of the enzymatic activity. The microbial cells typically remain intact, at least during some of the treatment, and intracellular enzymatic activity is not significantly adversely affected by the high pH treatment. In addition, the inventors have previously shown that microbial enzymes are more resistant to the high pH treatment in any case.
Accordingly, after the step of selective lysis of non-microorganism cells in the sample whilst retaining intact any microorganisms present in the sample, the method may comprise exposing the lysate to high pH conditions. The duration of exposure to the high pH conditions is typically less than 20 minutes and may be not more than 10, 9, 8, 7, 6 or 5 minutes and may be around 5, 6, 7, 8, 9 or 10 minutes. In some embodiments the treatment is carried out for between around 2 and 15 minutes, such as around 5 minutes. By “around” is meant plus or minus 30 seconds.
Any suitable reagent may be in order to provide high pH conditions. In particular embodiments, the high pH conditions comprise contacting the sample with an alkali or a buffer. In particular embodiments, NaOH or Na2CO3 is used. In specific embodiments, the concentration of the NaOH or Na2CO3 is around 5 mM or greater. The buffer may have a pKa value above 9. Examples of suitable buffers include borate, carbonate and pyrophosphate buffers.
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 polymerases. This is primarily due to the differential lysis conditions employed in the methods to ensure that only the non-microorganism enzymes are exposed to the high pH conditions. However, it may also be due to the greater resistance of microbial enzymes to these conditions. “High pH” is generally a pH of at least around 10, such as around 10, 11, 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. By “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 corresponding mammalian enzymes may be inactivated under the same pH conditions. This assists with the selective detection of microbial enzymatic activity in a sample containing both mammalian cells and microbial cells. In specific embodiments, 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 (Na2CO3). Such agents can readily be used, as shown herein, to increase the pH of the sample to high pH thus inactivating non-microorganism enzymatic activity whilst leaving the microbial (fungal and bacterial) enzymes 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 5 mM NaOH. In some embodiments, the alkali concentration is no more than 10 mM, such as 5, 6, 7, 8, 9 or 10 mM.
In further embodiments, 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). In specific embodiments, pH conditions may be increased to at least around 11, or at least 11.2. This treatment may, after a certain period of time, result in lysis of microorganisms in the sample and thus lead to nucleic acid modifying activity (e.g. polymerase and/or ligase) release into the sample. Thus, in some embodiments, the lysis of microorganisms is achieved by high pH treatment. This permits detection of nucleic acid modifying activity (e.g. polymerases and/or ligases) in the sample, originating from the microorganism, without the need for a separate cell lysis step. Under these conditions, mammalian ligases (such as blood ATP-dependent ligases) are inactivated. However, typically the methods include a separate step for lysing microorganisms in the sample, as discussed in greater detail herein.
In some embodiments, the treatment under high pH conditions is stopped by adding a reagent to lower the pH. This is done before the microorganisms are lysed. Suitable reagents include a buffer and/or an acid. Thus, the pH may be reduced by adding a neutralisation buffer. In specific embodiments, the buffer comprises a Tris-HCl buffer (e.g. pH 7.2 or 8). Other suitable agents for lowering the pH include acids such as hydrochloric acid (HCl) and sulphuric acid (H2SO4). These (and other) acids may be incorporated into a buffer as would be readily appreciated by one skilled in the art. One specific reagent useful for treating the sample after the pH has been elevated comprises a combination of Ammonium sulphate, Magnesium sulphate heptahydrate, Potassium chloride and Tris-HCl. More specifically, the reagent may comprise 10 mM Ammonium sulphate, 2 mM Magnesium sulphate heptahydrate, 10 mM Potassium chloride and 20 mM Tris-HCl [pH 8.0].
Step (b) may further comprise removing the non-microorganism cells from the particle-microorganism complexes e.g. by aspiration.
A “sample” in the context of the present invention is one which contains non-microorganism cells and in which it is desirable to test for the presence of a microorganism, such as a fungus (e.g. a yeast) and/or a bacterium, expressing nucleic acid modifying activity. Thus the sample may comprise, consist essentially of or consist of a clinical sample, such as a blood sample (to include whole blood, plasma, serum and blood containing samples, such as a blood culture or blood broth). The methods of the invention are particularly applicable to the rapid determination of negative (and positive) blood cultures. Thus, the sample may comprise a blood culture sample (or blood broth 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 10 ml, preferably a 1 ml blood culture sample.
The sample being used will depend on various factors, such as availability, convenience and the condition that is being tested for. Typical samples which may be used, but which are not intended to limit the invention, include whole blood, serum, plasma, platelet, joint fluid and urine samples etc. taken from a patient, most preferably a human patient. The patient may be suspected of suffering from, or being screened for, a bloodstream infection. The patient may be a hospitalised patient. The sample may be taken from a subject comprising more than 5, 10 or 15 million white blood cells (WBC) per ml of blood. The methods of the invention represent in vitro tests. They are carried out on a sample removed from a subject. However, in less preferred embodiments, the methods may additionally include the step of obtaining the sample from a subject. Methods of obtaining a suitable sample from a subject are well known in the art. Typically, however, the method may be carried out beginning with a sample that has already been isolated from the patient in a separate procedure. The methods will most preferably be carried out on a sample from a human, but the methods of the invention may have utility for many animals.
The methods of the invention may be used to complement any already available diagnostic techniques, potentially as a method of confirming an initial diagnosis. Alternatively, the methods may be used as a preliminary diagnosis method in their own right, since the methods provide a quick and convenient means of diagnosis. Furthermore, due to their inherent sensitivity, the methods of the invention require only a minimal sample, thus preventing unnecessary invasive surgery. Also, a large but non-concentrated sample may also be tested effectively according to the methods of the invention.
In specific embodiments according to all aspects of the invention, the microorganism that may be detected in the sample is a pathogenic microorganism, such as a pathogenic bacterium or fungus/yeast. The bacterium may be any bacterium which is capable of causing infection or disease in a subject, preferably a human subject. In one embodiment, the bacteria comprises or consists essentially of or consists of any one or more of Staphylococcus species, including Staphylococcus epidermidis and Staphylococcus aureus (and preferably methicillin resistant strains), Enterococcus species, Streptococcus species, Mycobacterium species, in particular Mycobacterium tuberculosis, Vibrio species, in particular Vibrio cholerae, Salmonella and/or Escherichia coli etc. The bacteria may comprise, consist essentially of or consist of Clostridium species and in particular C. difficile in certain embodiments. C. difficile is the major cause of antibiotic-associated diarrhoea and colitis, a healthcare associated intestinal infection that mostly affects elderly patients with other underlying diseases. Candida species such as C. albicans, C. parapsilosis and C. glabrata may be detected. Cryptococcus species such as C. neoformans may be detected. Fungaemia such as Candidaemia may be detected (presence or absence) using the invention. The microorganism is preferably (although this is not essential) indicated through its enzymatic activity. Thus, the methods provide an indication of viable, or recently so, microorganisms in the sample. After a period of time, if the microorganisms are not viable, the enzymatic activity would be lost from the sample. This represents an advantage of using enzymatic activity as an indicator of microorganisms in the sample over use of nucleic acid molecules, in particular DNA, which may persist for much longer.
The methods of the invention may involve identifying the nature of the infection, once the positive presence of a microorganism has been detected in the sample. Any suitable method may be employed for this further identification step.
The magnetic particles may be superparamagnetic particles.
The particles (e.g. magnetic particles) may have a greater affinity for the microorganisms than for the non-microorganism cells. The magnetic particles may bind to the microorganisms by non-specific binding.
The particles (e.g. magnetic particles) may have an outer polymeric surface that comprises polystyrene and/or poly(styrene/divinyl benzene).
The magnetic particles may comprise iron oxide. Preferably, the iron oxide is encapsulated by the outer polymeric surface. The particles may be an amalgam of iron oxide and polymer. The particles may be partially encapsulated by an outer polymeric surface. The polymer may comprise polystyrene,
The particles (e.g. magnetic particles) may have a diameter of between 0.05 and 1 μm, 0.1 and 0.5 μm, 0.2 and 0.3 μm. Preferably, the magnetic particles may have a diameter of between 0.2 and 0.3 μm. The particles (e.g. magnetic particles) may have a diameter of between 0.1 and 3 μm or 0.1 and 2 μm. More preferably, the particles have a diameter of between 0.1 and 1.0 μm.
The particles (e.g. magnetic particles) may have an outer polymeric surface. The outer polymeric surface of the magnetic particles may not be coated with any of (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from Apolipoprotein H protein, (iv) a Mannose Binding Lectin protein, (v) a polyamine or (vi) a cationic detergent.
The Mannose Binding Lectin (MBL) protein may be a genetically engineered protein based on MBL. For example, it may be a genetically engineered protein comprising the pathogen-binding portion of MBL fused to an Fc region of an immunoglobulin (i.e. FcMBL).
The outer surface (e.g. outer polymeric surface) of the particles (e.g. magnetic particles) may not be coated with any of (i) an antibody, (ii) a carbohydrate or (iii) an innate immune system protein.
The outer surface (e.g. outer polymeric surface) of the particles (e.g. magnetic particles) may not be coated with any of (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from Apolipoprotein H protein, (iv) Mannose Binding Lectin, or (v) a flocculating agent (e.g. a flocculating agent as defined in WO 03/102184).
The outer surface (e.g. outer polymeric surface) of the particle (e.g. magnetic particles) may not be coated with any of (i) an antibody, (ii) a carbohydrate, (iii) an innate immune system protein or (iv) a flocculating agent (e.g. a flocculating agent as defined in WO 03/102184).
The antibody may be a fragment or derivative of an antibody that retains antigen-specific binding function. Such fragments and derivatives include Fab fragments, ScFv, single domain antibodies, nanoantibodies, heavy chain antibodies etc.
The carbohydrate may be a monosaccharide, oligosaccharide (e.g. a disaccharide or a trisaccharide), a polysaccharide and/or a derivative thereof.
The outer surface (e.g. outer polymeric surface) of the particles (e.g. magnetic particles) may not be coated with a ligand. The outer surface (e.g. outer polymeric surface) of the particles (e.g. magnetic particles) may not be coated with a non-specific ligand (e.g. a non-specific ligand as described in WO01/53525). The outer surface (e.g. outer polymeric surface) of the particles (e.g. magnetic particles) may not be coated with a non-proteinaceous ligand (e.g. a non-proteinaceous ligand as described in WO01/53525).
The outer surface (e.g. outer polymeric surface) of the particles (e.g. magnetic particles) may be carboxylated. The outer surface (e.g. outer polymeric surface) of the particles (e.g. magnetic particles) may be coated only with carboxyl groups.
The outer surface (e.g. outer polymeric surface) of the particles (e.g. magnetic particles) may be coated with streptavidin. The outer surface (e.g. outer polymeric surface) of the particles (e.g. magnetic particles) may be coated with streptavidin and not coated with a ligand. The outer surface (e.g. outer polymeric surface) of the particles (e.g. magnetic particles) may be coated only with streptavidin.
The outer surface (e.g. outer polymeric surface) of the particles (e.g. magnetic particles) may not be coated.
The microorganism may be a pathogenic microorganism, optionally wherein the pathogenic microorganism is a pathogenic bacterium or fungus.
The non-microorganism cells may comprise red blood cells and/or white blood cells.
The invention further provides a composition. The composition provided herein may be for performing any of the methods described herein. All aspects and embodiments described in relation to the methods of the invention apply mutatis mutandis to the related compositions.
The composition may comprise: i) particles capable of forming complexes with microorganisms, wherein the particles have an outer surface; ii) sodium polyanethol sulfonate; and iii) at least one reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample.
The composition may comprise: i) particles capable of forming complexes with microorganisms, wherein the particles have an outer surface; ii) sodium polyanethol sulfonate; and iii) a detergent. A detergent is an example of a reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample.
The composition may further comprise microorganism cells and/or non-microorganism cells. The composition may comprise a sample suspected of containing microorganism cells and known to contain non-microorganism cells.
The composition may further comprise a buffer and/or sodium chloride.
In the composition, the reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample may be a detergent; optionally wherein the detergent is non-ionic. In the composition, the detergent may not be conjugated to the particles capable of forming complexes with microorganisms. Thus, typically the detergent forms part of a solution to which the particles are added and does not form part of the particles themselves.
In the composition, the particles may have a diameter of between 0.1 and 3 μm, or between 0.1 and 2 μm. Preferably, the particles have a diameter of between 0.1 and 1.0 μm
In the composition, the particles may be (and typically are) magnetic. In the methods, the particles may be superparamagnetic. The particles may comprise iron oxide. The iron oxide may comprise magnetite and/or maghemite. The iron oxide may not comprise a 1:1, 2:1, 3:1 or 4:1 ratio of Fe2+ and Fe3+.
In the composition, the outer surface of the particles capable of forming complexes with microorganisms may comprise a polymer; optionally the polymer may be carbon-based. The polymer may not comprise an inorganic polymer. The polymer may comprise polystyrene and/or poly(styrene/divinyl benzene).
In the composition, the outer surface of the particles capable of forming complexes with microorganisms may comprise or be coated with any one or more of: i) carboxylic acid groups; ii) amino groups; iii) hydrophobic groups; and iv) streptavidin; optionally the carboxylic acid groups; ii) amino groups; iii) hydrophobic groups may not be part of a polypeptide.
In the composition, the microorganism may be a pathogenic microorganism. For example, the pathogenic microorganism may be a pathogenic bacterium or fungus.
In the composition, the non-microorganism cells may comprise red blood cells and/or white blood cells.
In the composition, the sample may comprise blood, urine, saliva or milk, optionally wherein the sample is whole blood.
The invention further provides a kit for performing any of the methods described herein. All aspects and embodiments described in relation to the methods of the invention apply mutatis mutandis to the related kits.
The kit may comprise i) particles capable of forming complexes with microorganisms, wherein the particles have an outer surface; ii) sodium polyanethol sulfonate; and iii) at least one reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample.
The kit may comprise i) particles capable of forming complexes with microorganisms, wherein the particles have an outer surface; ii) sodium polyanethol sulfonate; and iii) a detergent. A detergent is an example of a reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample.
The detergent may be non-ionic. The detergent may not be conjugated to the particles capable of forming complexes with microorganisms. Thus, typically the detergent forms part of a solution to which the particles are added and does not form part of the particles themselves.
In the kit, the particles may have a diameter of between 0.1 and 3 μm or between 0.1 and 2 μm. Preferably, the particles have a diameter of between 0.1 and 1.0 μm
In the kit, the particles may be (and typically are) magnetic. The particles may be superparamagnetic. The particles may comprise iron oxide. The iron oxide may comprise magnetite and/or maghemite. The iron oxide may not comprise a 1:1, 2:1, 3:1 or 4:1 ratio of Fe2+ and Fe3+.
The outer surface of the particles may comprise a polymer; optionally the polymer may be carbon-based. The polymer may not comprise an inorganic polymer. The polymer may comprise polystyrene and/or poly(styrene/divinyl benzene).
In the methods, the outer surface of the particles capable of forming complexes with microorganisms may comprise or be coated with any one or more of: i) carboxylic acid groups; ii) amino groups; iii) hydrophobic groups; and iv) streptavidin; optionally the carboxylic acid groups; ii) amino groups; iii) hydrophobic groups may not be part of a polypeptide.
The kit may comprise: a) particles capable of forming complexes with microorganisms; b) sodium polyanethol sulfonate; c) at least one reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample; and d) detection means for detecting the absence or presence of microorganisms in the particle-microorganism complexes, wherein the detection means comprises a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the microorganisms, and wherein the nucleic acid molecule is at least partially double stranded and comprises uracil residues in the complementary strand.
The kit may comprise: a) particles capable of forming complexes with microorganisms; b) sodium polyanethol sulfonate; c) a detergent; and d) detection means for detecting the absence or presence of microorganisms in the particle-microorganism complexes, wherein the detection means comprises a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the microorganisms, and wherein the nucleic acid molecule is at least partially double stranded and comprises uracil residues in the complementary strand.
The kit may comprise: a) particles capable of forming complexes with microorganisms, wherein the particles have an outer surface that is not coated with any of (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from Apolipoprotein H protein, (iv) a Mannose Binding Lectin protein; and b) detection means for detecting the absence or presence of microorganisms in the particle-microorganism complexes, wherein the detection means comprises a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the microorganisms, and wherein the nucleic acid molecule is at least partially double stranded and comprises uracil residues in the complementary strand.
The kit may further comprise a reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample.
The kit may further comprise a buffer and/or sodium chloride.
In the kit, the reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample may be a detergent; optionally the detergent may be non-ionic. In the composition, the detergent may not be conjugated to the particles capable of forming complexes with microorganisms.
In the kit, the particles may have a diameter of between 0.1 and 3 μm, 0.1 and 2 μm. Preferably, the particles have a diameter of between 0.1 and 1.0 μm
In the kit, the particles may be magnetic. In the methods, the particles may be superparamagnetic.
In the kit, the outer surface of the particles capable of forming complexes with microorganisms may comprise a polymer; optionally the polymer may be carbon-based.
In the kit, the outer surface of the particles capable of forming complexes with microorganisms may comprise or be coated with any one or more of: i) carboxylic acid groups; ii) amino groups; iii) hydrophobic groups; and iv) streptavidin; optionally carboxylic acid groups; ii) amino groups; iii) hydrophobic groups may not be part of a polypeptide.
In the kit, the microorganism may be a pathogenic microorganism, optionally wherein the pathogenic microorganism may be a pathogenic bacterium or fungus.
In the kitn, the non-microorganism cells may comprise red blood cells and/or white blood cells.
In the kit, the sample may comprise blood, urine, saliva or milk, optionally wherein the sample is whole blood.
The kit may comprise (a) particles (e.g. magnetic particles) capable of forming complexes with microorganisms; and (b) detection means for detecting the absence or presence of microorganisms in the particle-microorganism complexes.
Any suitable detection means may be employed and they may represent the complete set of reagents needed for detecting the absence or presence of microorganisms in the particle-microorganism complexes.
In certain embodiments, the detection means comprises, or is, a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the microorganisms.
In some embodiments, the detection means comprise or further comprise reagents for nucleic acid amplification. The reagents for nucleic acid amplification may comprise a primer pair and/or at least one probe. In some embodiments, those primers and/or probes hybridise with a microorganism nucleic acid molecule. They may therefore allow detection of microorganisms in the sample by detecting the amplified microorganism nucleic acid molecule. Alternatively, the primers or probes hybridise to a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the microorganisms. Such nucleic acid molecules are described in further detail herein.
The kit may comprise: (a) particles (e.g. magnetic particles) capable of (selectively) forming complexes with microorganisms (i.e. particle-microorganism complexes); and (b) detection means for detecting the absence or presence of microorganisms in the particle-microorganism complexes. The detection means may comprise a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the microorganisms. The nucleic acid molecule may be at least partially double stranded and may, optionally, comprise uracil residues in the complementary strand. The complementary strand may comprise a base (e.g. dideoxyCytidine) at its 3′ end that blocks DNA polymerase-mediated extension of the second strand. The nucleic acid molecule may be any of the nucleic acid molecules described herein.
In the kits, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with any of (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from Apolipoprotein H protein, (iv) a Mannose Binding Lectin protein, (v) a polyamine or (vi) a cationic detergent.
In the kits, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with any of (i) an antibody, (ii) a carbohydrate or (iii) an innate immune system protein.
In the kits, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with any of (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from Apolipoprotein H protein, (iv) Mannose Binding Lectin, or (v) a flocculating agent (e.g. a flocculating agent as defined in WO 03/102184).
In the kits, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with any of (i) an antibody, (ii) a carbohydrate, (iii) an innate immune system protein or (iv) a flocculating agent (e.g. a flocculating agent as defined in WO 03/102184).
In the kits, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with a ligand.
In the kits, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is coated with streptavidin and is not coated with a ligand.
In the kits, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is coated only with streptavidin.
In the kits, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is coated only with carboxyl groups.
In the kits, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with any molecule or moiety capable of binding to a microorganism.
In the methods, the particles (e.g. magnetic particles) may have an outer surface (e.g. outer polymeric surface) that is not coated with any molecules or moieties.
The (substrate) nucleic acid molecule may be designed on the basis that the nucleic acid modifying enzyme comprises a DNA or RNA polymerase. In some embodiments, the DNA polymerase is DNA polymerase I. In additional or alternative embodiments, the nucleic acid modifying enzyme comprises a ligase, such as an ATP- or NAD-dependent ligase.
The detection means may further comprise reagents for nucleic acid amplification, optionally wherein the reagents for nucleic acid amplification comprise a primer pair and/or at least one probe that hybridises with the nucleic acid molecule.
The kit may further comprise a reagent capable of lysing microorganisms in the particle-microorganism complexes, optionally wherein the reagent capable of lysing microorganisms in the particle-microorganism complexes comprises the nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the microorganisms.
The kit may further comprise a reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample.
The reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample may comprise a combination of a detergent and one or more enzymes, wherein the one or more enzymes optionally comprise a proteinase and/or a DNAse. Suitable detergents and enzymes are discussed herein.
The kit may further comprise a high pH reagent e.g. a base or a buffer. This may, for example, be NaOH, e.g. 5 mM NaOH. Other suitable reagents are described herein.
The kit may further comprise a neutralisation buffer. The neutralisation buffer may be capable of restoring the pH of the sample following the high pH treatment. Suitable reagents are described herein.
The nucleic acid modifying enzyme may comprise: (a) a DNA or RNA polymerase, optionally wherein the DNA polymerase is DNA polymerase I; and/or (b) a ligase, optionally wherein the ligase is an ATP- and/or NAD-dependent ligase.
According to all relevant aspects and embodiments of the invention, the term “sodium polyanethol sulfonate” is intended to encompass all functionally equivalent derivatives and salt forms thereof (e.g. potassium polyanethol sulfonate, magnesium polyanethol sulfonate, etc.)
Throughout the disclosure, the term “particles” and “beads” may be used interchangeably.
The invention may also be defined by the following clauses:
The invention will be understood with respect to the following non-limiting examples:
In a manual format, two bead types (Merck Bio-Estapor (streptavidin-conjugated) 300 nm beads (Product—BE-M08/03; “Bio-Estapor”) and ademtech Bio-Adembeads Streptavidin Plus 200 nm beads (Product number 03222; “Bio-Ademtech”)) were compared to ApoH Technologies Peps6 beads (Reference—MP20006; “ApoH Peps6”).
In experiment 1A, an aliquot of Bio-Estapor beads (25 μL) and an aliquot of ApoH Pep6 beads (10 uL) were compared for binding. The higher volume of Bio-Estapor reflected the lower number of beads per mL in the material provided compared to the ApoH material. Three organisms were tested: E. coli (Gram negative bacterium), S. epidermidis (Gram positive bacterium) and C. albicans (yeast). 0.5 mL of organism suspension was exposed to the beads in 0.5 mL “TTGB” microbial binding buffer, provided in the ApoH Peps6 kit (“Peps6 Captobac”, Reference MP10031-50T).
After allowing the organism to bind for 30 min, the sample of beads was separated from the liquid supernatant by applying a magnetic field to concentrate the beads and removing the supernatant with a pipette. The beads were gently washed with three aliquots of wash buffer (50 mM Tris pH 8, 1% v/v Igepal CA-630, 150 mM NaCl, 0.25% v/v Tergitol 15-S-9) and the retained supernatant and the washed beads were analysed for viable organisms by two methods; colony counts on an Agar Petri dish and detection of microbial DNA by the enzymatic template generation and amplification (ETGA) test (as described in Zweitzig et al., 2012. Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes. Nucleic Acids Research 40, 14, e109, 1-12; and in WO2011/130584, WO2013/103744 and WO2016/005768).
The plate counts in Table 1A show that with the Bio-Estapor beads and E. coli, the great majority of growth is found from the beads (33 CFU) vs the supernatant (2 CFU) and this is similar to the result from ApoH Peps6. No growth was found with S. epidermidis and this organism did not appear to grow in the original broth. C. albicans showed approximately 10% of the CFU in the supernatant and 90% bound, for both Bio-Estapor and ApoH Peps6. These results indicate that the Bio-Estapor beads appear to bind organisms at an equivalent rate to the commercially available organism-binding beads Peps6 under the conditions of the test. The sensitive ETGA test supports the results but indicates that S. epidermidis may bind to Bio-Estapor better than Peps6 as shown by the lower Cq value.
E. coli
Staph
epidermidis
Candida
albicans
E. coli
S. epidermidis
C. albicans
E. coli
S. epidermidis
C. albicans
E. coli
s. epidermidis
c. albicans
Experiment 1B demonstrates E. coli binding under similar conditions to Experiment 1A although in 1B the wash steps were omitted. Experiment 1B shows that another bead, Bio-Ademtech, also binds organisms although at a lower level (see Table 1B). Here the plate counts indicate that approximately one third of the viable counts have bound to the bead. The more sensitive ETGA DNA polymerase assay indicates that half of the organisms remain on the beads, as the Cq for the beads and the supernatant are approximately equal.
In Experiment 2, ApoH Peps6, Bio-Estapor and Estapor beads with a carboxylated surface (Product MI-030/40; “Estapor COOH”) were compared. The number of organisms remaining in the supernatant after binding of E. coli to the beads for 30 min was measured using a fluorescent ATP assay (BacTiter-Glo Microbial Cell Viability Assay; Promega Corporation, G8230). Although this is an indirect test in that it does not directly detect the presence of organisms on the bead, it is a useful comparative test for the ligand-based beads (ApoH Peps6) and the non-ligand beads of the invention (Bio-Estapor and Estapor COOH). After binding of 1 mL of 104 CFU/mL E coli for 30 mL from a phosphate saline buffer, an aliquot of the supernatant was assayed for ATP as a measure of organism content using the BacTiter-Glo assay. The results in Table 2 show that the reduction in levels of organisms in the supernatant for Peps6 beads, Bio-Estapor and Estapor COOH were 33%, 27% and 24% respectively when measured using this technique.
Example 3 shows results from testing E. coli (EC), S. aureus (SA) and C. albicans (CA) in dilution series performed by automating the method for magnetic separation described in Example 1. The assay used Bio-Estapor 300 nm diameter beads as the capture medium with a binding buffer of TTGB containing 0.25% Tergitol. As 10-fold dilutions of each of the three organisms were made, so a continuous change in the Ct was recorded allowing a dose response curve to be constructed.
The following examples demonstrate the universal microbial capture of microorganisms by magnetic beads in Momentum's Magnitor test. The Magnitor test consists of two microbial detection read-outs:
Certain binding buffer components can improve microbial detection in the Magnitor assay, for example detergent-based lysis of blood.
Microbial binding performance was assessed for E. coli, S. aureus and C. albicans in a simple Tris+NaCl buffer (pH buffered with physiological salt conc. to prevent microbial osmotic shock that may occur in water only). A ‘no bead control’ sample-set was also included in this experiment to demonstrate that detection is dependent on the presence of magnetic beads for microbial capture.
Estapor beads (Merck, Cat #M1-30/40) washed 3×1 mL in 1× Tris+NaCl buffer: 40 μL beads resuspended in a final volume of 400 μL 1× Tris+NaCl buffer (1% solid content)
E. coli D1
E. coli D2
E. coli D3
E. coli D4
S. aureus D1
S. aureus D2
S. aureus D3
S. aureus D4
C. albicans D1
C. albicans D2
C. albicans D3
C. albicans D4
E. coli
S. aureus
C. albicans
ETGA ΔCt (averageNSC)
Critical Values Based on Average NSC (cfu/mL)
To compare two different blood lysis buffers in two different diluent formats for development of a simple and fast ‘Rapid Magnitor’ test (no wash step included in protocol).
Samples set up as above, and progressed immediately to Spin or Mag Beads protocol
Sample and dilutions of sample plated on COL plates (100 μL)
E. coli 1E−3
E. coli 1E−4
E. coli 1E−5
Sample source
Given the recent discovery that multiple bead types/sizes produce similar Magnitor results for microbial serial dilutions and NSCs, it was thought that a component within Momentum's binding buffer might be mediating/facilitating this observed universal microbial binding character. To investigate this possibility, a dilution series of E. coli was performed comparing the standard binding buffer (B-BUF) with a detergent-free B-BUF consisting of just Tris-HCl [pH8.0]+NaCl, to test whether the detergents in general are important for microbial binding. Sample-sets were prepared for blood-broth, broth-only and in 1× binding buffer only to compare results for different specimen types.
100 mL 10× Binding Buffers prepared fresh:
Estapor beads (Merck, Cat #M1-30/40) washed 3×1 mL in respective 1× buffer (diluted 10× B-BUF or 10× Tris+NaCl): 40 μL beads resuspended in a final volume of 400 μL 1× buffer (1% solid content)
Critical Values Based on Average NSC (cfu/mL)
To further investigate the importance of Momentum's binding buffer in mediating microbial binding the effect of pH buffering and salt on binding was investigated in a clean system (i.e. in the absence of any blood or broth).
25 mL of each buffer made fresh:
Estapor beads (Merck,Cat M1-30/40) washed 3×1 mL in respective 1× buffer (diluted 10× buffers): 30 μL beads resuspended in a final volume of 300 μL 1× buffer (1% solid content)
Protocol:
NB Dataset (i.e. No SPS)—Morning Experiment
E. coli 1e−3
E. coli 1e−4
E. coli 1e−5
E. coli 1e−6
All buffer (BUF-1 to 6) NC TVCs=0
E. coli 1e-3
E. coli 1e-4
E. coli 1e-5
E. coli 1e-6
E. coli 1e-3
E. coli 1e-4
E. coli 1e-5
E. coli 1e-6
PLUS Dataset (i.e with SPS)—Afternoon Experiment
E. coli 1e−3
E. coli 1e−4
E. coli 1e−5
E. coli 1e−6
All buffer (BUF-1 to 6) NC TVCs=0
E. coli 1e-3
E. coli 1e-4
E. coli 1e-5
E. coli 1e-6
E. coli 1e-3
E. coli 1e-4
E. coli 1e-5
E. coli 1e-6
E. coli 1e-3
E. coli 1e-4
E. coli 1e-5
E. coli 1e-6
E. coli 1e-3
E. coli 1e-4
E. coli 1e-5
E. coli 1e-6
E. coli 1e-3
E. coli 1e-4
E. coli 1e-5
E. coli 1e-6
To determine whether microbial capture and detection can occur when alternative blood lysis methods are employed.
Binding Buffers were prepared as follows:
BioEstapor beads (Merck, Cat #BE-M 08/0.3) were re-suspended prior to use.
S. aureus 1E−4
S. aureus 1E−5
S. aureus 1E−6
S. aureus 1E−4
S. aureus 1E−5
S. aureus 1E−6
S. aureus 1E−4
S. aureus 1E−5
S. aureus 1E−6
Critical Values Based on Average NSC (cfu/mL)
S. aureus 1E-4
S. aureus 1E-5
S. aureus 1E-6
S. aureus 1E-4
S. aureus 1E-5
S. aureus 1E-6
S. aureus 1E-4
S. aureus 1E-5
S. aureus 1E-6
However, microbial detection by ETGA is improved to differing extents by alternative blood lysis methods, due to effects on the reduction of blood-derived ETGA signal.
To determine the optimal SPS concentration for the Magnitor Rapid test using 1 mL whole blood samples. The secondary objective was to assess the effect of SPS on microbial viability in whole blood as determined by TVCs.
2×5 mL whole blood or BacTec PLUS aerobic blood broth (1:3 ratio) aliquoted for each sample-set (E. coli and NSC sample). Then SPS added as follows:
Then 5 μL of E. coli 1E-2 preculture added to each 5-mL specimen tube to recreate standard 1E-5 dilution sample
To compare alternative carboxylated magnetic beads of similar size using Momentum's Magnitor assay
E. coli and S. pyogenes o/n liquid cultures set up as standard in 3 mL broth and blood broth (BacTec PLUS aerobic) respectively, and incubated for 16-20 hours (37° C.)
The following day:
Internal Positivity Thresholds (Pt) calculated using NSCs (n=6) for each bead type: formula=PERCENTILE.INC(array,0.05)
E. coli
E. coli 1e-3
E. coli 1e-4
E. coli 1e-5
E. coli 1e-6
E. coli 1e-7
E. coli 1e-3
E. coli 1e-4
E. coli 1e-5
E. coli 1e-6
E. coli 1e-7
E. coli 1e-3
E. coli 1e-4
E. coli 1e-5
E. coli 1e-6
E. coli 1e-7
E. coli 1e-3
E. coli 1e-4
E. coli 1e-5
E. coli 1e-6
E. coli 1e-7
E. coli 1e-3
E. coli 1e-4
E. coli 1e-5
E. coli 1e-6
E. coli 1e-7
E. coli 1e-3
E. coli 1e-4
E. coli 1e-5
E. coli 1e-6
E. coli 1e-7
S. pyogenes
All carboxylated magnetic beads tested here demonstrate microbial binding as determined by ETGA and Confirm readouts. However, the sensitivity of microbial detection does vary somewhat, depending on the level of blood-derived ETGA signal and/or assay inhibition
To compare microbial capture performance for a variety of commercially-available magnetic beads of different size and functional coating using Momentum's Magnitor test (ETGA and Confirm technologies). Two experiments were performed to demonstrate microbial capture for automated (Protocol 1) and manual (Protocol 2) sample processing. Importantly, Protocol 2 included three bead resuspension washes to more convincingly demonstrate that ETGA/Confirm signal is specific to bead-bound microbial cells, rather than sample carryover (as opposed to Protocol 1, which included a single beads-magnetised wash step).
All beads washed in 1 mL 1× E-BUF (50 mM Tris-HCl [pH 8.0]+150 mM Sodium Chloride+1% Igepal+0.25% Tergitol) and resuspended to 1% solid in 1× E-BUF
Sample Set-Up (Performed Separately for Protocol 1 and 2 which were Performed on Different Days):
E. coli 1E-6
E. coli 1E-7
E. coli 1E-8
E. coli 1E-6
E. coli 1E-7
E. coli 1E-8
E. coli 1E-6
E. coli 1E-7
E. coli 1E-8
E. coli 1E−6
E. coli 1E−7
E. coli 1E−8
E. coli 1E−6
E. coli 1E−7
E. coli 1E−8
33.69†
31.78†
35.24†
E. coli 1E−6
E. coli 1E−7
E. coli 1E−8
These results demonstrate that a variety of different bead sizes and functional coatings produce comparable levels of microbial binding as determined by ETGA and Confirm readouts.
To compare microbial capture performance for a variety of commercially-available magnetic beads of different size and functional coating using Momentum's Magnitor test (ETGA and Confirm technologies). E. coli was tested previously (Bead size and coating I: source experiment: 20190221_WP7_Bead-Comparison_Analysis and 20190228_WP7_Bead-Comparison-3-wash_Analysis): to expand on this previous work, three additional microbial species were tested (S. aureus, S. pneumoniae and C. albicans).
All beads washed in 1 mL 1× Tris+NaCl and resuspended to 1% solid in 1× Tris+NaCl
S. aureus 1E−5
S. aureus 1E−6
S. aureus 1E−7
C. albicans 1E−2
C. albicans 1E−3
C. albicans 1E−4
S. pneumoniae 1E−2
S. pneumoniae 1E−3
S. pneumoniae 1E−4
S. aureus
S. aureus
S. aureus
C. albicans
C. albicans
C. albicans
S. pneumoniae
S. pneumoniae
S. pneumoniae
S. aureus 1E−5
S. aureus 1E−6
S. aureus 1E−7
C. albicans 1E−2
C. albicans 1E−3
C. albicans 1E−4
S. pneumoniae 1E−2
S. pneumoniae 1E−3
S. pneumoniae 1E−4
To compare microbial capture performance for a variety of commercially-available magnetic beads of different size and functional coating using Momentum's Magnitor test (ETGA and Confirm technologies). This experiment was performed using a simple buffer (50 mM Tris-HCl [pH 8.0]+150 mM NaCl) as the specimen and wash buffer i.e. no detergents used until the addition of microbial lysis mix.
All beads washed in 1 mL 1× Tris+NaCl (50 mM Tris-HCl [pH 8.0]+150 mM NaCl) and resuspended to 1% solid in 1× Tris+NaCl
E. coli 1E−6
E. coli 1E−7
E. coli 1E−8
S. aureus 1E−5
S. aureus 1E−6
S. aureus 1E−7
C. albicans 1E−2
C. albicans 1E−3
C. albicans 1E−4
E. coli 1E−6
E. coli 1E−7
E. coli 1E−8
S. aureus 1E−5
S. aureus 1E−6
S. aureus 1E−7
C. albicans 1E−2
C. albicans 1E−3
C. albicans 1E−4
E. coli 1E−6
E. coli 1E−7
E. coli 1E−8
S. aureus 1E−5
S. aureus 1E−6
S. aureus 1E−7
C. albicans 1E−2
C. albicans 1E−3
C. albicans 1E−4
To compare microbial capture performance for a variety of commercially-available magnetic beads of different size and functional coating using Momentum's Magnitor test (ETGA and Confirm technologies) in the absence of blood lysis i.e. no detergents in binding buffer
All beads washed in 1 mL 1× Tris+NaCl (50 mM Tris-HCl [pH 8.0]+150 mM NaCl) and resuspended to 1% solid in 1× Tris+NaCl
E. coli 1E−6
E. coli 1E−7
E. coli 1E−8
S. aureus 1E−5
S. aureus 1E−6
S. aureus 1E−7
C. albicans 1E−2
C. albicans 1E−3
C. albicans 1E−4
E. coli 1E−6
E. coli 1E−7
E. coli 1E−8
S. aureus 1E−5
S. aureus 1E−6
S. aureus 1E−7
C. albicans 1E−2
C. albicans 1E−3
C. albicans 1E−4
E. coli 1E−6
E. coli 1E−7
E. coli 1E−8
S. aureus 1E−5
S. aureus 1E−6
S. aureus 1E−7
C. albicans 1E−2
C. albicans 1E−3
C. albicans 1E−4
To investigate whether microbial capture using magnetic beads is possible for other complex biological fluids, in addition to blood
Plates and Liquid Cultures incubated overnight at 37° C.
E. coli D1
E. coli D2
E. coli D3
E. coli D4
E. coli D5
E. coli D1
E. coli D2
E. coli D3
E. coli D4
E. coli D5
E. coli D1
E. coli D2
E. coli D3
E. coli D4
E. coli D5
Note, no observable amplification in Candida channel for Confirm
E. coli D1
E. coli D2
E. coli D3
E. coli D4
E. coli D5
To show microbial capture and detection in non-blood specimens of milk and urine using non-lysing binding buffer and non-lysing wash buffer.
BioEstapor beads (Merck, Cat #BE-M 08/0.3) were re-suspended prior to use.
E. coli 1E-5
E. coli 1E-6
E. coli 1E-7
E. coli 1E-8
E. coli 1E-9
S. aureus 1E-5
S. aureus 1E-6
S. aureus 1E-7
S. aureus 1E-8
S. aureus 1E-9
C. albicans 1E-2
C. albicans 1E-3
C. albicans 1E-4
C. albicans 1E-5
C. albicans 1E-6
S. pneumoniae 1E-2
S. pneumoniae 1E-3
S. pneumoniae 1E-4
S. pneumoniae 1E-5
S. pneumoniae 1E-6
E. Coli 1E-5
E. Coli 1E-6
E. Coli 1E-7
E. Coli 1E-8
E. Coli 1E-9
S. aureus 1E-5
S. aureus 1E-6
S. aureus 1E-7
S. aureus 1E-8
S. aureus 1E-9
C. albicans 1E-2
C. albicans 1E-3
C. albicans 1E-4
C. albicans 1E-5
C. albicans 1E-6
S. pneumoniae 1E-2
S. pneumoniae 1E-3
S. pneumoniae 1E-4
S. pneumoniae 1E-5
S. pneumoniae 1E-6
E. Coli 1E-5
E. Coli 1E-7
E. Coli 1E-8
E. Coli 1E-9
S. aureus 1E-5
S. aureus 1E-6
S. aureus 1E-7
S. aureus 1E-8
S. aureus 1E-9
C. albicans 1E-2
C. albicans 1E-3
C. albicans 1E-4
C. albicans 1E-5
C. albicans 1E-6
S. pneumoniae 1E-2
S. pneumoniae 1E-3
S. pneumoniae 1E-4
S. pneumoniae 1E-5
S. pneumoniae 1E-6
NB: Pasteurised milk contains bacteria; these showed as a consistent ETGA Ct-22
E. Coli 1E−5
E. Coli 1E−6
E. Coli 1E−7
E. Coli 1E−8
E. Coli 1E−9
S. aureus 1E−5
S. aureus 1E−6
S. aureus 1E−7
S. aureus 1E−8
S. aureus 1E−9
C. albicans 1E−2
C. albicans 1E−3
C. albicans 1E−4
C. albicans 1E−5
C. albicans 1E−6
S. pneumoniae 1E−2
S. pneumoniae 1E−3
S. pneumoniae 1E−4
S. pneumoniae 1E−5
S. pneumoniae 1E−6
| Number | Date | Country | Kind |
|---|---|---|---|
| 1805479.1 | Apr 2018 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2019/050959 | 4/3/2019 | WO | 00 |
