WO2016005768A1 - Method and kit of detecting the absence of micro-organisms - Google Patents
Method and kit of detecting the absence of micro-organisms Download PDFInfo
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- WO2016005768A1 WO2016005768A1 PCT/GB2015/052006 GB2015052006W WO2016005768A1 WO 2016005768 A1 WO2016005768 A1 WO 2016005768A1 GB 2015052006 W GB2015052006 W GB 2015052006W WO 2016005768 A1 WO2016005768 A1 WO 2016005768A1
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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- C12Q2525/125—Modifications characterised by incorporating agents resulting in resistance to degradation
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- C12Q2533/00—Reactions characterised by the enzymatic reaction principle used
- C12Q2533/10—Reactions characterised by the enzymatic reaction principle used the purpose being to increase the length of an oligonucleotide strand
- C12Q2533/101—Primer extension
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- C12Q2535/113—Cycle sequencing
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- C12Q2545/00—Reactions characterised by their quantitative nature
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- G01N2333/91205—Phosphotransferases in general
- G01N2333/91245—Nucleotidyltransferases (2.7.7)
Definitions
- the present invention relates generally to the field of detecting the absence or presence of microorganisms in a sample.
- the methods typically rely upon measuring microbial enzyme activity (if any) present in a sample and may relate to such methods which are capable of being performed using nucleic acid amplification techniques such as the polymerase chain reaction.
- the methods of the invention therefore enable determination of the absence and presence of microbial pathogens in samples such as un-purified blood, blood culture and other body fluids.
- This invention also relates to reagents for use in such methods, and to test kits comprising such 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.
- WO96/002665 describes a method for determining the presence and/or amount of microorganisms and/or their intracellular material present in a sample characterized in that the amount of adenylate kinase in the sample is estimated by mixing it with adenosine diphosphate (ADP), determining the amount of adenosine triphosphate (ATP) produced by the sample from this ADP, and relating the amount of ATP so produced to the presence/or amount of adenylate kinase and to microorganisms and/or their intracellular material, wherein the conversion of ADP to ATP is carried out in the presence of magnesium ions at a molar concentration sufficient to allow maximal conversion of ADP to ATP.
- ADP adenosine diphosphate
- ATP adenosine triphosphate
- ligases in particular NAD-dependent ligases, are disclosed as a useful indicator of the presence of a (viable) microorganism in a sample.
- Ligases are enzymes which catalyze ligation of nucleic acid molecules. The ligation reaction requires either ATP or NAD+ as co-factor depending upon the ligase concerned.
- the use of NAD-dependent ligase activity is utilized as an indicator of the presence of a (viable) microorganism in a sample.
- WO201 1/130584 describes a method for detection of viable microorganisms based on detection of DNA or RNA polymerases in which a sample is contacted with a nucleic acid substrate that acts as a substrate for microbial polymerase, incubated under conditions suitable for polymerase activity from intact microorganisms and any resulting nucleic acid product is determined using a nucleic acid amplification technique such as quantitative polymerase chain reaction.
- a nucleic acid amplification technique such as quantitative polymerase chain reaction.
- WO2010/1 19270 describes a method for removing enzyme activity (in this case, DNA ligase) outside intact microorganisms and this can be used also for removal of contamination nucleic acid polymerase activity.
- Nucleic acid amplification assays may include an internal control probe to monitor for the correct functioning of the amplification reaction (see for example WO2013/103744 where this is applied to a DNA polymerase assay similar to that of WO201 1/130584). However, this internal control is added as part of the nucleic acid amplification reagent mix and would not detect prior nuclease activity.
- An additional problem with the method as described in WO201 1/130584 is a relative lack of sensitivity in detecting yeast such as C. albicans and C. glabrata.
- the art has focused on detection of the presence of microorganisms rather than determining their absence.
- determining their absence the applicants do not mean that the sample is necessarily sterile but may have an organism load that is sufficiently low as to be negative for practical purposes.
- blood cultures are often taken from patients suspected of having bloodstream infections which can be associated with sepsis, a condition that can be rapidly fatal if left untreated. It is routine for clinical microbiology laboratories to incubate such specimens for at least five days before reporting a negative result, during which time the patients are often kept on broad spectrum antibiotics. Typically up to 90% of such patients are negative, and so a large number of patients are left for 5 days on antibiotic therapy that is not necessary for their condition.
- a faster method for determining a negative result would be of significant value in reducing the cost of unnecessary antibiotic therapy and provide health benefits in terms of reducing the risks of C. difficile infection, antibiotic toxicity and lowering the rate of increase in antimicrobial resistance.
- the inventors have devised and tested a range of improvements to existing ETGA assays with a view to optimising determination of the absence or presence of a microorganism in a sample.
- the foundation of the invention is thus methods of detecting the absence or presence of a micro-organism in a sample comprising:
- the invention provides a method of detecting the absence or presence of a micro-organism in a sample comprising:
- nucleic acid molecule specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism, characterised in that the nucleic acid molecule is modified so as to protect it from nuclease activity.
- the 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 contacted with the sample in step (a).
- 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 as shown herein, 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.
- the synthetic nucleotides comprise
- the synthetic nucleotides are phosphorothioate nucleotides.
- the synthetic nucleotides replace at least one up to all of the nucleotides in the nucleic acid molecule.
- the invention provides a method of detecting the absence or presence of a micro-organism in a sample comprising:
- nucleic acid molecule specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism, characterised in that the nucleic acid molecule is added to the sample at a concentration of at least 2nM but less than 50nM.
- the nucleic acid substrate has been utilised at a
- step (a) of contacting the sample with the substrate nucleic acid molecule typically involves addition of the substrate in a lysis mixture which lyses the microorganisms if present in the sample. Further details of the lysis reagent/mixture are provided herein.
- the methods of the invention comprise adding to the sample deoxyribonucleotide triphosphates (dNTPs) at a concentration of more than 50 ⁇ , such as 55 to 300 ⁇ , or 60 to 250 ⁇ , or 75 to 200 ⁇ in particular at least 10 ⁇ .
- the dNTPs may be added in either step (a) and/or step (b) in some embodiments.
- the concentration as stated herein, is typically the concentration in the lysis mixture used to lyse the micro-organisms if present in the sample.
- step (a) of contacting the sample with the substrate nucleic acid molecule typically involves addition of the substrate in a lysis mixture which lyses the micro-organisms if present in the sample, in which the lysis mixture contains the dNTPs. Further details of the lysis reagent/mixture are provided herein.
- the invention provides a method of detecting the absence or presence of a micro-organism in a sample, the sample containing a non-microorganism source of nucleic acid modifying activity comprising: (a) treating the sample under high pH conditions for no more than 8 minutes in order to inhibit the non-microorganism source of nucleic acid modifying activity (whilst not affecting the nucleic acid modifying activity of the microorganism in the sample), (b) contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample,
- the duration of the high pH conditions is 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.
- the treatment is carried out for between around 2 and 15 minutes, such as around 5 minutes.
- around is meant plus or minus 30 seconds.
- any suitable reagent may be added to the sample in order to provide high pH conditions.
- the high pH conditions comprise contacting the sample with an alkali.
- NaOH or Na2C03 is used.
- the concentration of the NaOH or Na2C03 is around 5mM or greater.
- the high pH conditions typically inhibit the activity of nucleic acid modifying enzymes including ATP-dependent ligase and polymerases from non-microorganism sources such as mammalian cells, but do not inhibit the activity of the microbial ligases or
- High pH is generally a pH of at least around 10, such as around 10, 1 1 , 12, 13 or 14.
- Low pH is generally a pH of less than or equal to around 4, such as around 4, 3, 2, or 1 .
- around is meant 0.5 of a pH unit either side of the stated value. Altering the pH of the sample may be achieved using any suitable means, as would be readily appreciated by one skilled in the art.
- Microbial enzymes such as polymerases and ligases may be resistant to extremes of pH, whereas mammalian ligases may be inactivated under the same pH conditions. This permits selective detection of microbial ligases in a sample containing both mammalian cells and microbial cells.
- the conditions that inhibit the activity of non-microorganism nucleic acid modifying activity, such as ATP-dependent ligase, from mammalian cells but which do not inhibit the activity of the microorganism source of nucleic acid modifying activity, such as microbial ligases comprise treating the sample with sodium hydroxide (NaOH) or sodium carbonate (Na2C03).
- Such agents can readily be used, as shown herein, to increase the pH of the sample to high pH thus inactivating mammalian ligase activity whilst leaving the microbial (fungal and bacterial) ligases active.
- Suitable concentrations and volumes of the appropriate agent can be applied by a skilled person.
- the NaOH is at least around 5mM NaOH.
- the alkali concentration is no more than 10mM, such as 5, 6, 7, 8, 9 or 10mM.
- the pH is around 12 to inactivate mammalian nucleic acid modifying activity (such as polymerase and/or ATP-dependent ligase activity), but not microbial nucleic acid modifying activity (such as polymerase and/or ligase activity).
- pH conditions may be increased to at least around 1 1 , or at least
- This treatment may result in lysis of micro-organisms in the sample and thus lead to nucleic acid modifying activity (e.g. polymerase and/or ligase) release into the sample.
- nucleic acid modifying activity e.g. polymerase and/or ligase
- mammalian ligases such as blood ATP-dependent ligases
- the methods include a separate step for lysing microorganisms in the sample, as discussed in greater detail herein below.
- the treatment under high pH conditions is stopped by adding a reagent to lower the pH.
- Suitable reagents include a buffer and/or an acid.
- the buffer comprises a Tris-HCI buffer (e.g. pH 7.2 or 8).
- suitable agents for lowering the pH include acids such as hydrochloric acid (HCI) and sulphuric acid (H2S04). These (and other) acids may be incorporated into a buffer as would be readily appreciated by one skilled in the art. These steps may be incorporated into step (a) of the method outlined above.
- step (a) is performed at a temperature between around (to mean plus or minus 0.5 degrees) 15 and 30 degrees Celsius. In certain embodiments, step (a) is performed at room temperature. The entirety of the methods described herein may be performed at these temperatures.
- the invention further provides a method of detecting the absence or presence of a micro-organism in a sample, the sample containing a non-micro-organism source of nucleic acid modifying activity comprising:
- Step (ii) is an optional step because, in some embodiments, the lysed cell material does not need to be separated from the intact microorganisms. This is because step (iii) is used to inhibit the nucleic acid modifying activity found in the lysed cell material in any case.
- lysed cell material is meant the product of lysis of the non-microorganisms. This includes the cell membranes and intracellular content of the lysed cells.
- the methods may comprise the steps of:
- step (x) specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism.
- step (iii) or (iv) respectively, or the entire method is performed at a temperature between 1 5 and 30 degrees Celsius.
- step (iii) or (iv) respectively or the entire method may be performed at room temperature.
- the reagent that lyses non-micro-organisms, in particular mammalian cells, if present in the sample but does not lyse micro-organisms in the sample may be any suitable reagent.
- 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 8.5g/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-micro-organisms, in particular mammalian cells, if present in the sample but no (or insignificant) lysis of micro-organisms 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 1 5 and 30 degrees Celsius or at room temperature.
- separation of the lysed cell material from the intact microorganisms (if any) in the sample may be performed by any suitable method. It may for example rely upon a form of affinity purification, such as an (polyclonal) antibody-based approach. It may rely upon filtration in some embodiments. Separation may rely upon centrifugation of the sample to form a pellet containing micro-organisms if present in the sample.
- Centrifugation of the sample may be performed at any suitable speed and for any suitable duration.
- the sample may be centrifuged at a speed of between 3000 and 1 0000g, such as around 7000g or 7300g.
- the sample may be centrifuged for a suitable period of time to ensure successful lysis of non-micro-organisms, in particular mammalian cells, if present in the sample but no or insignificant lysis of micro-organisms in the sample. This may be determined in conjunction with the speed of centrifugation.
- the time period may be between around 1 and 30 minutes, such as 1 , 2, 3, 4, 5, 10, 1 5, 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.
- the lysed cell material may be discarded (in the form of a supernatant) and the non-lysed cells retained (for example as a pellet).
- the high pH reagent comprises NaOH or Na2C03.
- the concentration of the high pH reagent is around 5mM or greater.
- the pH lowering reagent comprises a buffer or an acid, such as a Tris-HCI buffer.
- the buffer may be a pH 7.2 or 8 buffer in specific embodiments.
- any microorganisms in the sample are separated from the pH modifying conditions. This may be achieved by a second centrifugation of the sample to form a pellet containing micro-organisms if present in the sample, followed by removal of the supernatant from the pellet. Suitable centrifugation conditions are discussed above.
- the method then requires lysis of any separated microorganisms to permit detection of nucleic acid modifying activity. This 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 modifying activity within the cells.
- the components may be selected from carrier/serum proteins such as BSA,
- the lysis mixture of the invention may include the following components:
- Serum protein such as albumin (e.g. BSA)
- a suitable lysis mixture is set forth below in table 1 and forms a separate aspect of the invention: Table 1 . Lysis mixture components
- Lysis may also require disruption of the cells.
- the cells may be disrupted using the lysis mixture in combination with physical and/or enzymatic means.
- 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.
- Enzymatic disruption may require use of an agent selected from lysostaphin, lysozyme and/or lyticase in some embodiments.
- the step of contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample may include adding the nucleic acid molecule to the lysis mixture.
- the sample is then incubated under conditions suitable for nucleic acid modifying activity. This may involve incubation at an optimum temperature for nucleic acid modifying activity.
- the sample may be incubated at a temperature between around 15 and 40 degrees Celsius, such as around 37 degrees Celsius. This may be for any suitable period of time, for example between 5 and 60 minutes, such as around 5, 10, 15, 20, 25 or 30 minutes.
- the nucleic acid modifying activity may be inactivated prior to the modified nucleic acid molecule detection step. This may be achieved by elevating the temperature, for example to a temperature above 60 degrees Celsius, such as 95 degrees Celsius for a suitable time period. This may be a relatively short time period such as 1 , 2, 3, 4, 5, 10, 15 or more minutes.
- determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism may be performed by any suitable method as discussed herein.
- Preferred methods are nucleic acid amplification based and may permit quantification of the nucleic acid modifying activity (and thus microorganisms) in the sample.
- the inventors have also investigated the use of an internal positive control (IPC) molecule in the context of the ETGA methods.
- the invention may rely upon inclusion of the IPC with the substrate nucleic acid molecule so that the IPC is exposed to identical conditions. They have found that residual nuclease activity in the sample may affect the substrate added to the sample (particularly in the lysis mixture, as defined herein). Thus, there is an advantage in protecting the IPC from nuclease activity.
- the invention also provides a method of detecting the absence or presence of a micro-organism in a (liquid) sample, the sample potentially containing a non-micro- organism source of nuclease activity comprising:
- Separation in step (ii) is an optional step because, in some embodiments, the lysed cell material does not need to be separated from the intact microorganisms.
- an alternative or additional step of inactivating the nucleic acid modifying activity found in the lysed cell material is performed. Any suitable inactivation technique may be employed as discussed herein. For example, inactivation may be of nucleic acid modifying activity and/or nuclease activity in the lysed cell material. Inactivation may be achieved using any suitable means, for example high pH treatment as discussed herein. The fact that the microorganisms remain intact may protect them from an inactivation treatment.
- the invention also provides a method of detecting the absence or presence of a micro-organism in a (liquid) sample, the sample potentially containing a non-microorganism source of nuclease activity comprising:
- the protected IPC is particularly advantageous in the context of use of protected substrate molecules.
- the (substrate) nucleic acid molecule is also modified so as to protect it from nuclease activity. This ensures that both nucleic acid molecules are protected and subjected to the same conditions. Any suitable means may be employed in order to protect the nucleic acid molecules from nuclease activity. Non-limiting examples include incorporation of methylation into the nucleic acid molecules, 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.
- the synthetic nucleotides are phosphorothioate nucleotides.
- the synthetic nucleotides replace at least one up to all of the nucleotides in the nucleic acid molecules.
- the IPC and substrate nucleic acid molecule are modified in the same manner. This is with a view to providing as equal as possible protection from nuclease activity.
- the IPC is modified so as to protect it from nuclease activity
- the IPC is pre-modified so as to protect it from nuclease activity i.e. the IPC is modified so as to protect it from nuclease activity before it is contacted with the sample.
- the invention also contemplates using the IPC in order to monitor potential
- the invention also provides a method of detecting the absence or presence of a micro-organism in a (liquid) sample, the sample potentially containing a non-micro-organism source of nuclease activity comprising:
- step (vi) specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism, characterised in that the IPC nucleic acid molecule is susceptible to nuclease activity and is used to identify contaminating nuclease activity in the pellet.
- Separation in step (ii) is an optional step because, in some embodiments, the lysed cell material does not need to be separated from the intact microorganisms.
- an alternative or additional step of inactivating the nucleic acid modifying activity found in the lysed cell material is performed. Any suitable inactivation technique may be employed as discussed herein.
- inactivation may be of nucleic acid modifying activity and/or nuclease activity in the lysed cell material. Inactivation may be achieved using any suitable means, for example high pH treatment as discussed herein. The fact that the microorganisms remain intact may protect them from an inactivation treatment.
- a method of detecting the absence or presence of a micro-organism in a (liquid) sample, the sample potentially containing a non-microorganism source of nuclease activity comprising:
- 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.
- UDG Uracil DNA Glycosylase
- the first strand of the partially double stranded (substrate) nucleic acid molecule comprises (or consists of) synthetic nucleotides (e.g.
- the double stranded region encompasses the 3' end regions of the first and second (complementary) strands.
- 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 micro-organism 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).
- 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.
- UDG Uracil DNA Glycosylase
- 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 micro-organism in the sample.
- the IPC nucleic acid molecule comprises identical primer binding sites to the nucleic acid molecule such that there is competition for primer binding (in step (vi) or (f) of the method).
- a nucleic acid probe is added (in step (vi) or (f)) which binds to a target probe sequence within the nucleic acid molecule.
- the probe binds to the sense strand of the nucleic acid molecule.
- a further nucleic acid probe is added in step (vi) or (f) respectively which binds to a target probe sequence within the IPC nucleic acid molecule.
- the nucleic acid probe does not bind to the IPC nucleic acid molecule and the further nucleic acid probe does not bind to the nucleic acid molecule.
- the nucleic acid probe and/or further nucleic acid probe may be labelled. Preferably, they are differently labelled.
- the complementary strand of the nucleic acid molecule comprises a modification at the 3' end to prevent extension.
- This modification may comprise incorporation of a non-extendible nucleotide.
- the non-extendible nucleotide is a dideoxy nucleotide triphosphate (ddNTP), such as dideoxyCytidine.
- the (substrate) nucleic acid molecule may be modified so as to protect it from nuclease activity. Suitable modifications are discussed herein and may be selected from incorporation of methylation, protection of the 3' and/or 5' ends, incorporation of synthetic nucleotides.
- Examples of synthetic nucleotides comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides.
- the synthetic nucleotides are phosphorothioate nucleotides.
- the invention further provides a method of detecting the absence or presence of a micro-organism in a sample, the sample containing a non-micro-organism source of nucleic acid modifying activity comprising:
- nucleic acid molecule specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the microorganism, wherein the nucleic acid molecule is modified so as to protect it from nuclease activity.
- Step (ii) is an optional step because, in some embodiments, the lysed cell material does not need to be separated from the intact microorganisms. This is because step (iii) is used to inhibit the nucleic acid modifying activity found in the lysed cell material in any case.
- the invention further provides a method of detecting the absence or presence of a microorganism in a sample, the sample containing a non-microorganism source of nucleic acid modifying activity comprising:
- nucleic acid molecule specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism, wherein the nucleic acid molecule is modified so as to protect it from nuclease activity.
- the modification may be selected from incorporation of methylation, protection of the 3' and/or 5' ends, incorporation of synthetic nucleotides.
- the synthetic nucleotides may comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides.
- the synthetic nucleotides are phosphorothioate nucleotides.
- the nucleic acid molecule is added to the sample at a concentration of at least 2nM and less than 50nM (e.g. 2nM to 25nM, 5nM to 15nM, or 7.5 to 12.5nM), such as 2nM, 5nM, 7.5nM or 10nM.
- the nucleic acid molecule may be included in the lysis mixture used in step (vi) or (h) of the method (i.e. the specified concentration is the concentration in the lysis mixture).
- steps (vi) and (vii) or (h) and (i) respectively may effectively be combined as a single step in some embodiments.
- the lysis mixture may be as specified in Table 1 or as discussed elsewhere in this disclosure.
- the method may similarly comprise adding to the sample deoxyribonucleotide triphosphates at a concentration of more than 50 ⁇ , preferably at least 100 ⁇ , such as 55 to 300 ⁇ , or 60 to 250 ⁇ , or 75 to 200 ⁇ .
- the dNTPs may be included in the lysis mixture used in step (vi) or (h) of the method (i.e. the specified concentration is the concentration in the lysis mixture).
- steps (vi) and (vii) or (h) and (i) respectively may effectively be combined as a single step in some embodiments.
- the high pH reagent may be or comprise NaOH or Na2C03. In specific embodiments, the concentration of the high pH reagent is around 5mM or greater.
- the pH lowering reagent may comprise a buffer or an acid, such as a Tris-HCI buffer (e.g. pH 7.2 or 8).
- a Tris-HCI buffer e.g. pH 7.2 or 8
- step (iii) or (d) is performed at a temperature between 15 and 30 degrees Celsius or is performed at room temperature.
- Each and/or all steps of the method may be performed at a temperature between 15 and 30 degrees Celsius or at room temperature in some embodiments. Where nucleic acid amplification steps such as PCR are utilised, those steps will need to be performed at appropriate temperatures as detailed herein and understood by the skilled person.
- a nuclease susceptible IPC may be employed.
- step (vii) or (i) respectively may comprise contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample together with an internal positive control (IPC) nucleic acid molecule, wherein the IPC nucleic acid molecule is susceptible to nuclease activity and is used to identify contaminating nuclease activity in the pellet.
- IPC internal positive control
- step (vii) or (i) respectively may comprise contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample together with an internal positive control (IPC) nucleic acid molecule, wherein the IPC nucleic acid molecule is modified so as to protect it from nuclease activity.
- IPC internal positive control
- the synthetic nucleotides may be or comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides.
- the synthetic nucleotides are phosphorothioate nucleotides. If both molecules are modified, it is preferable that they are modified in the same or similar manner such that the nuclease resistance is comparable. This permits the IPC to perform a most useful comparator role to determine the impact of nuclease activity on the substrate molecule. As already mentioned, in some embodiments, steps (vi) and (vii) or (h) and (i) are performed together.
- the nucleic acid molecule is added to the sample together with a lysis reagent to form a lysis mixture.
- the detection of the modified nucleic acid molecule may be detected by a range of methods including by sequencing or nucleic acid amplification.
- step (ix) or (k) respectively comprises a nucleic acid amplification step. Any suitable nucleic acid molecule may be employed.
- the nucleic acid molecule incorporates uracil residues.
- the nucleic acid molecule is at least partially double stranded and comprises uracil residues in the complementary strand.
- the methods may comprise adding Uracil DNA Glycosylase (UDG) to the sample in order to degrade the uracil residues in the complementary strand.
- UDG Uracil DNA Glycosylase
- the complementary strand of the nucleic acid molecule comprises a modification at the 3' end to prevent extension.
- extension is meant the addition of further nucleotides. Any suitable modification may be employed.
- the modification is or comprises incorporation of a non-extendible nucleotide.
- a non-extendible nucleotide Any suitable non-extendible nucleotide may be employed.
- the non-extendible nucleotide may be or comprise a dideoxy nucleotide triphosphate (ddNTP), such as dideoxyCytidine.
- ddNTP dideoxy nucleotide triphosphate
- the IPC nucleic acid molecule comprises identical primer binding sites to the nucleic acid molecule such that there is competition for primer binding during the step of detection of the modified nucleic acid molecule (in step (ix) or (k)).
- the methods include use of a probe, in particular in step (ix) or (k).
- a nucleic acid probe is added in step (ix) or (k) of the method. This probe binds to a target probe sequence within the (sense strand of the) nucleic acid molecule.
- binds is meant hybridization under the conditions applied to the method as would be readily appreciated by one skilled in the art.
- a further nucleic acid probe is utilised, for example added in step (ix) or (k), which binds to a target probe sequence within the IPC nucleic acid molecule.
- the nucleic acid probe does not bind to the IPC nucleic acid molecule and the further nucleic acid probe does not bind to the (substrate) nucleic acid molecule.
- the probes and nucleic acid molecules (IPC or substrate) can be designed to avoid unwanted cross- hybridization using techniques and tools (such as online design tools) known in the art.
- the nucleic acid probe and/or further nucleic acid probe may be labelled.
- the nucleic acid probe and further nucleic acid probe are differently labelled.
- they may be labelled with fluorophores which have different wavelengths of maximal emission. Suitable pairs of labels can be readily selected by one skilled in the art, for example FAM and Texas Red may be used as different labels.
- the nucleic acid modifying activity may be any activity that is useful for indicating microorganism viability.
- the nucleic acid modifying activity is an enzymatic activity provided by the microorganism. Examples include polymerase and/or ligase activity.
- the nucleic acid modifying activity is polymerase activity.
- Polymerase activity may comprise DNA and/or RNA polymerase activity.
- the polymerase activity is DNA and/or RNA polymerase activity.
- Ligase activity may be ATP or NAD dependent.
- Other nucleic acid modifying activities relevant to viability may alternatively be measured such as phosphatase, kinase and/or nuclease activity.
- the action of the nucleic acid modifying activity on the substrate nucleic acid molecule produces an extended nucleic acid molecule.
- Suitable substrate molecules are described herein in detail. Reference can also be made to WO201 1/130584, WO2010/1 19270 and WO2009/007719 (the pertinent disclosures of which are hereby incorporated) where suitable substrate molecules useful for detecting nucleic acid modifying activity are disclosed. In the case of phosphatase activity, suitable nucleic acid molecules are disclosed in WO2006/123154, which disclosure is hereby incorporated by reference.
- the substrate nucleic acid molecules for use in the methods, and inclusion in the kits, of the invention must be of sequence and structure such that the NAD-dependent ligase can act on the molecule to produce a detectable ligated (novel) nucleic acid molecule.
- Suitable substrate nucleic acid molecules for use in the invention are described in more detail in the experimental section below.
- the substrate may be comprised of the following molecules:
- the nucleic acid molecule is partially double stranded and comprises uracil residues in the complementary strand. This permits Uracil DNA Glycosylase (UDG) to degrade the uracil residues in the complementary strand following extension and thus prevents the substrate molecule from being non-specifically amplified in the absence of extension (i.e. in the absence of nucleic acid modifying activity in the sample).
- the complementary strand of the nucleic acid molecule comprises a modification at the 3' end to prevent extension.
- extension is meant the addition of further nucleotides. Any suitable modification may be employed.
- the modification is or comprises incorporation of a non-extendible nucleotide.
- a non-extendible nucleotide Any suitable non-extendible nucleotide may be employed.
- the non-extendible nucleotide may be or comprise a dideoxy nucleotide triphosphate
- ddNTP such as dideoxyCytidine as shown in SED ID NO: 6.
- variants of these sequences may be utilised in the present invention.
- additional flanking sequences may be added.
- Alternative ddNTPs may be employed.
- Variant sequences may have at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide sequence identity with the nucleotide sequences of the substrate nucleic acid molecules set forth as SEQ ID NOs 6 and 7.
- the nucleic acid molecules may incorporate synthetic nucleotide analogues as appropriate or may be RNA or PNA based for example, or mixtures thereof. Suitable modifications, for example, to protect from nuclease activity, are described herein. They may be labelled, such as using a fluorescent label, or FRET pair, in certain embodiments to facilitate detection. Suitable detection methods are described herein.
- the (substrate) nucleic acid molecules include any natural nucleic acid and natural or synthetic analogues that are capable of being acted upon by nucleic acid modifying activity in order to generate 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 is present in excess, and in particular in large molar excess, over the nucleic acid modifying activity (provided by the microorganisms) in the sample.
- the nucleic acid modifying activity provided by the microorganisms
- the substrate and/or primers may incorporate complementary non-naturally occurring molecules which can base pair with each other, to avoid nonspecific detection of genomic DNA.
- pyDAD and puADA may be incorporated into primers and substrate molecules as appropriate (Sismour et al.,
- the methods of the invention may incorporate an IPC molecule.
- Any suitable IPC may be employed according to the requirements of the method.
- the following IPC may be used in the invention: gcc gat ate gga caa egg ccg aac tgg gaa ggc gag ate age agg cca cac gtt aaa gac aga gag aca aca acg ctg gcc gtt tgt cac cga cgc eta (SEQ ID NO: 3)
- SEQ ID NO: 3 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.
- 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
- nick displacement technology and nick displacement amplification (WO 2004/067726).
- the list above is not intended to be exhaustive. Any nucleic acid amplification technique may be used provided the appropriate nucleic acid product is specifically amplified. Similarly, sequencing based methodologies may be employed in some embodiments to include any of the range of next generation sequencing platforms. Amplification is achieved with the use of amplification primers specific for the sequence of the modified nucleic acid molecule which is to be detected. In order to provide specificity for the nucleic acid molecules primer binding sites corresponding to a suitable region of the sequence may be selected.
- nucleic acid molecules may also include sequences other than primer binding sites which are required for detection of the novel nucleic acid molecule produced by the modifying activity in the sample, for example RNA Polymerase binding sites or promoter sequences may be required for isothermal amplification technologies, such as NASBA, 3SR and TMA.
- 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.
- 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).
- Suitable primers for use in the methods of the invention are set forth in the experimental section below. They include primers comprising, consisting essentially of or consisting of SEQ ID NO: 4 and/or 5. These primers form a separate aspect of the invention. It is noted that variants of these sequences may be utilised in the present invention. In particular, additional sequence specific flanking sequences may be added, for example to improve binding specificity, as required. Variant sequences may have at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide sequence identity with the nucleotide sequences of the primers set forth in the experimental section.
- the primers may incorporate synthetic nucleotide analogues as appropriate or may be RNA or PNA based for example, or mixtures thereof.
- the primers may be labelled, such as with fluorescent labels and/or FRET pairs, depending upon the mode of detection employed. Probes may be utilised, again which may be labelled, as desired.
- the methods of the invention are carried out using nucleic acid amplification techniques in order to detect the 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.
- 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. Realtime and end-point techniques are advantageous because they keep the reaction in a "single tube". This means there is no need for downstream analysis in order to obtain results, leading to more rapidly obtained results.
- Real-time and end-point quantitation of PCR reactions may be accomplished using the TaqMan® system (Applied Biosystems), see Holland et al; Detection of specific polymerase chain reaction product by utilising the 5'-3' exonuclease activity of Thermus aquaticus DNA polymerase; Proc. Natl. Acad. Sci. USA 88, 7276-7280 (1991 ), Gelmini et al. Quantitative polymerase chain reaction-based homogeneous assay with flurogenic probes to measure C-Erb-2 oncogene amplification. Clin. Chem. 43, 752-758 (1997) and Livak et al. Towards fully automated genome wide polymorphism screening. Nat. Genet.
- This type of probe may be generically referred to as a hydrolytic probe.
- Suitable hydrolytic/Taqman probes for use in real time or end point detection are also provided.
- the probe may be suitably labelled, for example using the labels detailed below.
- 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.
- Suitable probes useful in the present invention are set forth as SEQ ID NO: 1 and 2.
- 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 1 7, 804 - 807 (01 Aug 1999).
- Additional real-time or end-point detection techniques which are well known to those skilled in the art and which are commercially available include Lightcycler® technology, Amplifluour® primer technology, DzyNA primers (Todd et al., Clinical Chemistry 46:5, 625-630 (2000)), or the PlexorTM qPCR and qRT-PCR Systems.
- the products of nucleic acid amplification are detected using real-time or end point techniques.
- the real-time technique consists of using any one of hydrolytic probes (the Taqman® system), FRET probes (Lightcycler® system), hairpin primers (Amplifluour® system), hairpin probes (the Molecular beacons system), hairpin probes incorporated into a primer (the Scorpion® probe system), primers incorporating the complementary sequence of a DNAzyme and a cleavable fluorescent DNAzyme substrate (DzYNA), Plexor qPCR and oligonucleotide blocking systems.
- 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
- Amplification products may be quantified to give an approximation of the microbial nucleic acid modifying activity in the sample and thus the level of microorganisms in the sample.
- "absence or presence” is intended to encompass quantification of the levels of microorganisms in the sample.
- the reaction mixture will contain all of; the sample under test, the substrate nucleic acid molecule(s), reagents, buffers and enzymes required for amplification of the modified nucleic acid molecule optionally in addition to the reagents required to allow real time or end-point detection of amplification products.
- the entire detection method for the nucleic acid modifying activity may occur in a single reaction, with a quantitative output, and without the need for any intermediate washing steps.
- single tube reaction is advantageous because there is no need for downstream analysis in order to obtain results, leading to more rapidly obtained results. Furthermore keeping the reaction in a "single tube” environment reduces the risk of cross
- the methods of the invention may be carried out in step-wise fashion.
- a first step it may first be necessary to prepare the sample in a form suitable for use in the method of the invention.
- selective cell lysis or increasing cellular permeability may be required.
- Capture of specific nucleic acid modifying activity, such as polymerase or ligase may also be desirable again as described herein.
- Other (sources of) nucleic acid modifying activity, such as nuclease activity may be inhibited etc.
- the steps of the methods of the invention prior to the amplification step may not comprise a step performed at a temperature of more than 40 ° C, more than 50 ° C, more than 60 ° C, more than 70 ° C, more than 80 ° C, more than 90 ° C or more than 95 ° C.
- the methods of the invention may not comprise any steps performed at a temperature of more than 40 ° C, more than 50 ° C, more than 60 ° C, more than 70 ° C, more than 80 ° C, more than 90 ° C or more than 95 ° C.
- the steps of the methods of the invention prior to the amplification step may be performed at a temperature between 10 and 50 degrees Celsius, between 15 and 45 degrees Celsius, between 20 and 40 degrees Celsius, between 25 and 40 degrees Celsius, between 30 and 40 degrees Celsius, between 25 and 35 degrees Celsius, or between 15 and 30 degrees Celsius, optionally the steps of the methods prior to the amplification step may be performed at room temperature.
- steps of the methods of the invention may all be performed at a temperature between 10 and 50 degrees Celsius, between 15 and 45 degrees Celsius, between 20 and 40 degrees Celsius, between 25 and 40 degrees Celsius, between 30 and 40 degrees Celsius, between 25 and 35 degrees Celsius, or between 15 and 30 degrees Celsius, optionally all of the steps of the methods may be performed at room temperature.
- the methods of the invention may comprise a step of inactivating nuclease activity in the sample.
- the methods of the invention do not comprise a step of inactivating nuclease activity in the sample. If performed, the step of inactivating nuclease activity takes place after the incubation step and before the step of specifically determining the absence or presence of a modified nucleic acid molecule (e.g. by amplification).
- a "sample” in the context of the present invention is defined to include any sample in which it is desirable to test for the presence of a microorganism, such as a fungus (e.g. a yeast) or a bacterium, expressing nucleic acid modifying activity.
- the sample may comprise, consist essentially of or consist of a clinical sample, such as a blood sample.
- the methods of the invention are particularly applicable to the rapid determination of negative blood cultures.
- the sample may comprise a blood culture sample from a patient suspected of suffering from, or being screened for, a bloodstream infection.
- the sample may be any suitable volume such as 1 to 10ml, preferably a 1 ml blood culture sample.
- sample may be or comprise an in vitro assay system for example.
- Samples may comprise, consist essentially of or consist of beverage or food samples or preparations thereof, or pharmaceutical or cosmetic products such as personal care products including shampoos, conditioners, moisturisers etc., all of which are tested for microbial contamination as a matter of routine.
- the sample may comprise, consist essentially of or consist of tissue or cells and may comprise, consist essentially of or consist of a sputum or a blood sample or a platelet sample for example.
- the methods and kits of the invention may be used to monitor contamination of surfaces, such as for example in locations where food is being prepared. Contamination is indicated by the presence of microbial nucleic acid modifying activity.
- the contamination may be from any microbial source, in particular bacterial or fungal (e.g yeast) contamination.
- the invention is also useful in monitoring environmental conditions such as water supplies, wastewater, marine environments etc.
- the invention is also useful in monitoring bacterial growth in fermentation procedures and in air sampling where bacteria or spore content can be assessed in hospital, industrial facilities or in biodefence applications.
- the methods of the invention have various utilities in addition to screening samples for the absence or presence of a microorganism. Accordingly, in a further aspect the invention provides for use of a method as described herein for screening for resistance of a micro-organism to an agent directed against micro-organism.
- the method may involve steps of exposing the sample containing the microorganism of interest to the agent and then performing a method of the invention to determine whether the microorganism is resistant. If the microorganism is resistant, the modified nucleic acid molecule will be detected. Typically such methods are performed using well
- the invention provides for use of a method as described herein for screening candidate agents which may be capable of killing or preventing growth of one or more micro-organisms.
- This method may involve exposing the sample containing the microorganism to the agent and then performing a method of the invention. If the agent is an effective killing agent, there would be no (or reduced) modified nucleic acid detected.
- Such methods are performed using well characterised samples, such as a cultured clinical isolate of a microorganism of interest.
- the methods may be performed as a time course experiment to determine whether the agent is able to prevent growth of the microorganism (even if not able to kill). There may be a parallel reaction run in the absence of the agent to determine the growth of the microorganism in the absence of the agent. This provides a comparison for the effectiveness of the agent in terms of growth inhibition activity.
- the invention provides for use of the method as described herein for diagnosing an infection, or a disease associated with the presence of a micro-organism in a subject.
- sample will generally be a clinical sample.
- the sample being used will depend on 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 and urine samples etc. taken from a patient, most preferably a human patient.
- the test will be an in vitro test carried out on a sample removed from a subject.
- the above-described diagnostic methods may additionally include the step of obtaining the sample from a subject.
- the method may be carried out beginning with a sample that has already been isolated from the patient in a separate procedure.
- the diagnostic methods will most preferably be carried out on a sample from a human, but the method of the invention may have diagnostic utility for many animals.
- the diagnostic methods of the invention may be used to complement any already available diagnostic techniques, potentially as a method of confirming an initial diagnosis.
- 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.
- the diagnostic methods of the invention require only a minimal sample, thus preventing unnecessary invasive surgery.
- a large but non-concentrated sample may also be tested effectively according to the methods of the invention.
- the methods of the invention have multiple applications beyond detection of contaminating organisms in a sample.
- the description provided above with respect to the various aspects of the invention applies mutatis mutandis to the other aspects of the invention and is not repeated for reasons of conciseness.
- suitable controls may be incorporated for each method of the invention.
- the microorganism is a pathogenic microorganism, such as a pathogenic bacterium.
- the bacterium may be any bacterium which is capable of causing infection or disease in a subject, preferably a human subject.
- the bacteria comprises or consists essentially of or consists of any one or more of
- Staphylococcus species in particular 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.
- the molecule which is being tested in the method is an antimicrobial compound.
- any molecule may be tested. Examples include antimicrobial agents, nucleic acid molecules including siRNA (dsRNA) molecules and antisense molecules, small molecules, antibodies and all derivatives thereof including Fab fragments, variable region fragments and single domain antibodies for example provided they retain binding affinity etc.
- the method may be carried out in a high throughput context to screen large numbers of molecules in a short period of time.
- the antimicrobial agent in one embodiment, may be taken from the two main types of antimicrobial agents, antibiotics (natural substances produced by micro-organisms) and chemotherapeutic agents (chemically synthesized), or may be a hybrid of the two such as semi-synthetic antibiotics (a subsequently modified naturally produced antibiotic) or synthetic antibiotics (synthesised versions of natural antibiotics).
- Suitable candidate antimicrobial agents may, following a positive result in the methods of the invention in terms of ability to kill or prevent growth of a bacterium or bacterial cell or other suitable micro-organism be tested for at least one or more of the following properties:
- the agent should be non-toxic to the subject and without adverse side effects
- the agent should be non-allergenic to the subject
- the agent should preferably be cheap and readily available/easy to manufacture
- the agent should be sufficiently potent that pathogen resistance does not develop (to any appreciable degree). This feature may be tested according to the methods described above.
- a combination of multiple suitable antimicrobial agents may be tested for ability to treat an infection and/or for resistance thereto.
- Antibiotics or derivatives thereof which may be tested for resistance and perhaps also for their novel ability to treat certain infections may be selected from the following groups, provided by way of example and not limitation; beta-lactams such as penicillin, in particular penicillin G or V, and cephalosporins such as cephalothin, semi-synthetic penicillins such as ampicillin, methicillin and amoxicillin, clavulanic acid preferably used in conjunction with a semi-synthetic penicillin preparation (such as clavamox or augmentin for example), monobactams such as aztreonam, carboxypenems such as imipenem, aminoglycosides such as streptomycin, kanamycin, tobramycin and gentamicin, glycopeptides such as vancomycin, lincomycin and clindamycin, macrolides such as erythromycin and oleandomycin, polypeptides such as polymyxin and bacitracin, polyenes such as amphotericin
- the invention provides for use of a method as described herein for detecting the presence of microorganism contamination in a platelet containing sample.
- the methods may incorporate sub-steps of:
- the invention also relates to kits useful in performing the methods of the invention.
- kits for carrying out a method as described herein comprising: (a) at least one nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, wherein the at least one nucleic acid molecule is at least partially double stranded and comprises uracil residues in the complementary strand, characterised in that the nucleic acid molecule is modified so as to protect it from nuclease activity
- kits may incorporate any of the components required to perform the methods of the invention. Accordingly, all discussion of the methods of the invention applies mutatis mutandis.
- the nucleic acid molecule is pre-modified so as to protect it from nuclease activity.
- the kit further comprises a nucleic acid probe which binds to a target probe sequence within the (sense strand of the) nucleic acid molecule.
- the kit may further comprise a further nucleic acid probe which binds to a target probe sequence within the IPC nucleic acid molecule.
- the nucleic acid probe does not bind to the IPC nucleic acid molecule and the further nucleic acid probe does not bind to the nucleic acid molecule.
- the nucleic acid probe and/or further nucleic acid probe may be labelled.
- the nucleic acid probe and further nucleic acid probe are differently labelled. For example, they may be labelled with fluorophores which have different wavelengths of maximal emission.
- Suitable pairs of labels can be readily selected by one skilled in the art, for example FAM and Texas Red may be used as different labels.
- the complementary strand of the nucleic acid molecule comprises a modification at the 3' end to prevent extension.
- extension is meant the addition of further nucleotides.
- Any suitable modification may be employed.
- the modification is or comprises incorporation of a non-extendible nucleotide.
- Any suitable non-extendible nucleotide may be employed.
- the non-extendible nucleotide may be or comprise a dideoxy nucleotide triphosphate
- ddNTP such as dideoxyCytidine
- the IPC is modified so as to protect it from nuclease activity.
- the IPC is pre-modified so as to protect it from nuclease activity. Suitable modifications are discussed in greater detail herein and may be selected from incorporation of methylation, protection of the 3' and/or 5' ends, incorporation of synthetic nucleotides.
- the synthetic nucleotides may be or comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides.
- the synthetic nucleotides are phosphorothioate nucleotides. If both molecules are modified, it is preferable that they are modified in the same or similar manner such that the nuclease resistance is comparable.
- the kit further comprises a high pH reagent.
- the high pH reagent may be or comprise NaOH or Na2C03. In specific embodiments, the concentration of the high pH reagent is around 5mM or greater.
- the kit may further comprise a pH lowering agent.
- the pH lowering reagent may comprise a buffer or an acid, such as a Tris-HCI buffer (e.g. pH 7.2 or 8).
- kits may incorporate a suitable carrier in which the reactions take place.
- such a carrier may comprise a multi-well plate, such as a 48 or 96 well plate for example.
- a carrier allows the detection methods to be carried out in relatively small volumes - thus facilitating scale up and minimising the sample volume required.
- kits will typically incorporate suitable instructions. These instructions permit the methods of the invention to be carried out reliably using the kits of the invention.
- the invention may be further defined in the following set of numbered clauses:
- a method of detecting the absence or presence of a micro-organism in a sample comprising:
- a method of detecting the absence or presence of a micro-organism in a sample comprising:
- nucleic acid molecule specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism, characterised in that the nucleic acid molecule is added to the sample at a concentration of at least 2nM but less than 50nM.
- step (a) and/or (b) comprises adding to the sample deoxyribonucleotide triphosphates at a concentration of at least 100 ⁇ .
- a method of detecting the absence or presence of a micro-organism in a sample, the sample containing a non-micro-organism source of nucleic acid modifying activity comprising:
- step (a) is performed at a temperature between 15 and 30 degrees Celsius.
- step (a) is performed at room temperature. 14. The method of any one of clauses 1 to 13 wherein the method is performed at a temperature between 15 and 30 degrees Celsius.
- a method of detecting the absence or presence of a micro-organism in a sample, the sample containing a non-micro-organism source of nucleic acid modifying activity comprising:
- (x) specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism.
- step (a)(iii) or (b)(iv) is performed at a temperature between 1 5 and 30 degrees Celsius.
- step (a)(iii) or (b)(iv) is performed at room temperature.
- a method of detecting the absence or presence of a micro-organism in a (liquid) sample, the sample potentially containing a non-micro-organism source of nuclease activity comprising:
- a method of detecting the absence or presence of a micro-organism in a (liquid) sample, the sample potentially containing a non-micro-organism source of nuclease activity comprising:
- nucleic acid molecule 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.
- UDG Uracil DNA Glycosylase
- a method of detecting the absence or presence of a microorganism in a sample, the sample containing a non-microorganism source of nucleic acid modifying activity comprising:
- nucleic acid molecule specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism, wherein the nucleic acid molecule is modified so as to protect it from nuclease activity. or (a) incubation of the sample with a reagent that lyses non-micro-organisms if present in the sample but does not lyse micro-organisms in the sample
- step (vii) or (i) respectively comprises adding to the sample deoxyribonucleotide triphosphates at a concentration of at least 100 ⁇ .
- step (vii) or (i) respectively comprises adding to the sample deoxyribonucleotide triphosphates at a concentration of at least 100 ⁇ .
- the high pH reagent comprises NaOH or Na2C03.
- step (iv) or (d) respectively is performed at a temperature between 15 and 30 degrees celcius.
- step (iv) or (d) respectively is performed at room temperature.
- step (vi) or (i) respectively comprises contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample together with an internal positive control (IPC) nucleic acid molecule, wherein the IPC nucleic acid molecule is susceptible to nuclease activity and is used to identify contaminating nuclease activity in the pellet.
- IPC internal positive control
- step (vi) or (i) respectively comprises contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample together with an internal positive control (IPC) nucleic acid molecule, wherein the IPC nucleic acid molecule is modified so as to protect it from nuclease activity.
- IPC internal positive control
- step (k) comprises adding Uracil DNA Glycosylase (UDG) to the sample in order to degrade the uracil residues in the complementary strand.
- UDG Uracil DNA Glycosylase
- a kit for carrying a method according to any one of clauses 1 to 84 comprising :
- nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, wherein the at least one nucleic acid molecule is at least partially double stranded and comprises uracil residues in the complementary strand, characterised in that the nucleic acid molecule is modified so as to protect it from nuclease activity
- IPC internal positive control
- kit of clause 85 wherein the kit further comprises a nucleic acid probe which binds to a target probe sequence within the (sense strand of the) nucleic acid molecule.
- kit of clause 85 or 86 wherein the kit further comprises a further nucleic acid probe which binds to a target probe sequence within the IPC nucleic acid molecule.
- the high pH reagent comprises NaOH or Na2C03.
- FIG 1 Improved ETGA detection of microorganisms by increasing dNTP and substrate concentration.
- Each chart shows the ct value obtained for the detection of the ETGA target substrate (FAM channel) in ETGA detection experiments for a range of relevant microorganisms.
- the negative blood culture control was >39.9 ct units
- the negative reagent negative controls were >40 ct units
- the positive reagent controls were ⁇ 20 ct units.
- Figure 2 Detection of IPC molecule in a blood culture sample prepared with IPC DNA added only in the microbial Lysis mixture (LM) or PCR mastermix (MM) compared to a negative control sample.
- LM microbial Lysis mixture
- MM PCR mastermix
- IPC DNA was added to the LM to provide the same ct value as when added to the MM.
- Data shows that there is a complete loss in detection of the IPC molecule (in a 40 cycle PCR reaction) in the negative blood culture sample compared to the negative control (with no blood) when added to LM and not when added to MM.
- Figure 3 Increased background caused by adding IPC to LM instead of MM.
- the data plotted on the chart shows ct value in the FAM channel (detecting ETGA substrate) versus total viable count (TVC) obtained for blood culture samples using a protocol where the IPC had been added to LM (diamonds) and MM (squares).
- the amount of IPC added to LM was equivalent to 50x higher than in MM for each PCR reaction.
- the measured background was higher when using 50x the normal concentration of IPC in LM compared to using the standard concentration of IPC in MM. Background levels were measured by an ETGA test procedure with IPC in the MM (blue dashed line) or LM
- FIG. 4 ETGA test background reduction and improved test sensitivity.
- Graphs show fluorescence detected in the FAM channel in qPCR reaction from ETGA tests carried out on a dilution series of C. albicans in blood culture.
- the qPCR reaction contained a FAM- labelled probe, capable of detecting the modified ETGA substrate, so amplification indicates the presence of a microorganism.
- Fig. 4a shows the results of a set of ETGA tests carried out using standard substrate and IPC oligos.
- Fig. 4b shows the results obtained from exactly the same samples using PTO substrate and IPC. Note that background is much lower in Fig. 4b, and that it is possible to detect lower number of yeast cells in the test.
- Figure 5 Improvement of yeast detection by use of PTO oligos.
- Graph shows sensitivity of detection of yeast (C. albicans) in an ETGA test using standard oligos compared to an ETGA test using PTO oligos.
- FIG. 6 Detection of less robust microorganisms by ETGA. Chart shows how detection of a delicate strain of H. influenzae is affected by the ETGA test procedure. Pure culture of S. aureus and H. influenzae (10 5 cfu) was added to the general test protocol (1 0 mL) at different stages. Data shows that detection was significantly reduced when
- microorganisms are added before the NaOH resuspension step.
- FIG. 7 Controlling exposure to NaOH to improve detection of H. influenzae.
- Graph shows the effect of controlling the amount of time that a culture sample containing 10 5 cfu H. influenzae is exposed to NaOH in the ETGA test procedure compared to the standard procedure.
- the general protocol for 10 mL was carried out on a suspension of H. influenzae in BacT/ALERT broth without blood; after resuspension in NaOH and incubation for 0, 0.5, 2.5 and 5 min, 1 mL 200mM Tris-HCI [pH7.2] was added prior to centrifugation.
- Figure 8 Improving the 1 ml ETGA protocol with a pH lowering step.
- Blood culture samples containing A) no spike, B) H. influenzae (10 5 cfu), C) H. influenzae (1 0 4 cfu), D) S. aureus (1 0 5 cfu), E) S. aureus (1 0 4 cfu) were tested with the original 1 ml procedure (based on a resuspension in 1 mL NaOH) and a procedure containing a pH-lowering step (resuspension in 0.75 mL NaOH, 5 min incubation, 0.5 mL Reagent C).
- the lower ct value indicates that the microorganisms are detected more strongly.
- FIG. 9A-C Cognitor Minus results for E. coli spiked blood broth samples and the positive control (Pol(+)). Data is shown for Cognitor Minus samples analysed at time 0, 2 and 20 hours with or without the 95° C step for (A)experiment 2, (B) experiment 3 and (C) experiment 4. Data for experiment 1 is not shown because samples were only analysed by QPCR at time 0 hours.
- Reagent B 5 mM NaOH
- the remaining pellet was resuspended in 0.5 mL of Reagent C and immediately transferred to a new tube containing a mixture of glass beads ( 0.1 mm and 0.5mm glass beads; supplied by CamBio cat 131 18-400, and 131 16-400 respectively). A further centrifugation was carried out in order to pellet any suspended cells with the glass beads, and again, the supernatant was removed and discarded.
- microbial Lysis Mixture containing the ETGA substrate (LM; containing reagents L1 , L2, L3 at a ratio of 7:2:1 , see Table 1 ) was added to the glass beads and placed in a Disruptor Genie (Scientific Industries, Inc.) cell disruptor for 6 min at 2800 rpm to lyse microbial cells. After disruption, samples were placed in a 37°C heating block and incubated for 20 min, then transferred to another heating block at 95°C and incubated for 5 min. After incubation, samples were cooled to room temperature whilst the PCR reagents were prepared.
- LM containing reagents L1 , L2, L3 at a ratio of 7:2:1 , see Table 1
- PCR mastermix containing a general Taq polymerase PCR mastermix (Roche - cat 04902343001 ), primers for the ETGA substrate, internal positive control - IPC - DNA, FAM-labelled probe for the ETGA substrate, Texas Red labelled probe for the IPC, and (1 .2ul) UDG enzyme (Bioline - cat no BIO-27044)) in a SmartCycler PCR tube (Cepheid). Samples were placed in the SmartCycler PCR and subjected to the following reaction conditions; 1 cycle; 40°C 10 min, 50°C 10 min, 95°C 5 min
- Fam labelled probe (a molecular beacon):
- the substrate components are;
- the general protocol was modified by increasing the amount of ETGA substrate in LM by 10-fold (from 0.001 ⁇ to 0.01 ⁇ ) and increasing the amount of dNTP 2-fold (from 50 ⁇ to 100 ⁇ ).
- the increased quantity of substrate and dNTP enabled improved detection of C. albicans (Fig. 1 a), E. coli (Fig. 1 b), S. aureus (Fig. 1 c), E. faecalis (Fig. 1 d), P. aeruginosa (Fig. 1 e), or 2 different strains of H. influenzae (including a delicate clinical strain) (Fig. 1 f and Fig. 1 g). Data is shown in Figure 1 .
- IPC IPC molecule at the same time as the ETGA substrate molecule was thought to be an improvement on the original protocol. If IPC was added in LM, the IPC would be subject to exactly the same test conditions as the ETGA substrate and therefore provide a more accurate test control. For example, conditions that may negatively impact the substrate molecule such as nuclease activity that could digest the substrate, would also affect the IPC. If the IPC is added later (in MM for example) it would not be subject to the same conditions and may result in false interpretation of the data. If IPC is added at the same time as the ETGA substrate the magnitude of the effect of the test conditions on the nucleic acid templates could be measured.
- Figure 2 shows that test conditions could have a negative impact on the detection of the ETGA substrate; when adding comparable amounts of IPC DNA in LM rather than in MM, it was found that there was indeed a loss in the ability to detect the IPC in blood culture specimens compared to negative control samples (without blood). The same loss of detection was not seen when using IPC in MM. Loss of the IPC when added in LM was attributed to nucleases that may have been active during the 37 °C incubation step of the ETGA protocol. Nucleases were most likely to have originated from the blood specimen.
- the ETGA substrate molecule could also be lost.
- DNA targets IPC and ETGA substrate
- DNA can be protected from nuclease activity by various means, by modification (e.g. methylation, end modification) or using non-standard nucleotides during the synthesis of synthetic oligonucleotides (e.g. locked nucleic acids, phopsphorothioate nucleotides).
- ETGA test has been found to be less sensitive to yeasts than bacteria. The reason for this not known but is likely to be due to a combination of reason such as, differences in in vitro activity or absolute quantity of the fungal enzymes in cells compared to the bacterial enzymes, or sensitivity to inhibitors.
- ETGA substrate was added to LM at 0.01 ⁇ and PTO-modified IPC was added at a sufficient quantity to achieve a ct value of 37-41 in a 50 cycle PCR reaction.
- a dilution series of yeast cells (10 5 , 10 4 , 10 3 and 0 cfu/ml) in blood culture was tested with the original ETGA test protocol with standard oligos and with PTO modified oligos. Both tests were run on exactly the same spiked blood cultures (Figure 4). Data showed that the ETGA test with standard oligos yielded results that included high levels of background that may have occluded the detection of low levels of yeast cells, whereas results obtained with PTO modified oligos did not suffer from the same effect.
- results showed that PTO oligos reduced the overall background to undetectable levels on the same culture specimens, thus allowing the lowering of the threshold level in the qPCR reaction and potentially increasing the sensitivity of the ETGA test.
- the threshold level was set at 50 units due to the amount of background fluorescence detected, but, when using the PTO oligos the threshold level could be lowered to 10 units, or lower if required.
- PTO oligos were detected later in the PCR reaction than when using standard oligos, but the PTO qPCR reaction could also be run for longer (50 cycles rather than 40) because the level of background was so low.
- microcentrifuge tube containing a mixture of glass beads. A further centrifugation for 3 min at 7300 x g was carried out in order to pellet any suspended cells with the glass beads, and again, the supernatant was removed and discarded.
- microbial Lysis Mixture (LM; containing ETGA substrate see table 1 in Example 1 above) was added to the glass beads and placed in a Disruptor Genie cell disruptor for 6 min at 2800 rpm to lyse microbial cells. After disruption, samples were placed in a 37 °C heating block and incubated for 20 min, then tansferred to another heating block at 95 °C and incubated for 5 min. After incubation, samples were cooled to room
- PCR mastermix MM; containing a general Taq polymerase PCR mastermix, primers for the ETGA substrate, internal positive control - IPC - DNA, FAM-labelled probe for the ETGA substrate, Texas Red labelled probe for the IPC, and UDG enzyme
- the remaining pellet was resuspended in 0.5 mL of Reagent C (1 .32g/L ammonium sulphate, 0.49 g/L magnesium sulphate heptahydrate, 0.75 g/L potassium chloride, 20 mM Tris-HCI, pH8.0) and immediately transferred to a new tube containing a mixture of glass beads. A further centrifugation was carried out in order to pellet any suspended cells with the glass beads, and again, the supernatant was removed and discarded.
- Reagent C (1 .32g/L ammonium sulphate, 0.49 g/L magnesium sulphate heptahydrate, 0.75 g/L potassium chloride, 20 mM Tris-HCI, pH8.0
- a single clinical microbial isolate, identified as Haemophilus influenzae gave a false negative result in the ETGA test during a clinical performance evaluation.
- microorganism was detected by standard automated blood culture in Biomerieux
- the time taken to centrifuge the sample increases the total time that the sample is exposed to NaOH by 8 min. Shortening the length of time could be achieved and controlled by neutralisation of the alkali, or, at least lowering the pH of the sample after an optimal period of incubation time by adding 1 mL of 200 mM Tris-HCI buffer, pH 7.2 to the NaOH.
- Results demonstrate that neutralisation (or significant lowering the pH) of the NaOH lead to lower ct values (from identical samples) and therefore improved sensitivity of detection (see Figure 7).
- Data also suggested that the shorter incubation time would be better, but again, short incubation times did not allow sufficient removal of contaminants to enable reliable PCR amplification, leading to reaction failure or high background levels and false positive results.
- Lowering of the pH of the sample after NaOH treatment could potentially be achieved by adding any suitable buffer or acid.
- the preferred method of lowering the pH would be to use Reagent C (a Tris-HCI buffer, pH 8) because the reagent is already used in the test.
- the purpose of the work outlined in this example was to assess the effect of the 95°C step on the performance of the Cognitor Minus test.
- the Cognitor Minus test was carried out using bacteria-spiked blood broth samples with or without the 95 °C step to compare the following characteristics:
- the earlier steps in the Cognitor Minus test aim to lyse blood cells and wash away blood- derived proteins such as DNA polymerases, which will produce non-microorganism derived ETGA template DNA, and nuclease enzymes which may digest microorganism- derived ETGA template DNA. Any resulting intact microorganisms are then lysed by the addition of lysis mix (LM) and bead milling. Following microorganism lysis and the ETGA reaction, samples contain a mixture of microorganism proteins, LM components, newly synthesised ETGA template DNA and residual blood cell proteins.
- LM lysis mix
- the 95°C step is intended to denature all proteins in order to protect the ETGA template DNA and internal process control (IPC) DNA from nuclease digestion so that it can be successfully detected by QPCR.
- IPC internal process control
- the Cogritor Minus test was carried out using bacteria-spiked blood cultures with or without the 95°C step. The samples were analysed by QPCR at three time points: immediately after sample preparation; after 2 hours at room temperature; and after a further 18 hours at 4°C. The aim was to compare the Ct values obtained and the consistency of Ct values across the three time points as an indicator of ETGA template DNA stability. An additional experiment was performed using LM containing UMO ETGA substrate to test whether the stability of the resulting ETGA template DNA differs from that of LM containing PTO ETGA substrate. Materials and methods
- Reagent A 5% (w/v) Saponin, 5% (v/v) Tween 20 and 146 mM Sodium chloride
- Reagent B 5 mM Sodium hydroxide.
- Reagent C 10 mM Ammonium sulphate, 2 mM Magnesium sulphate heptahydrate, 10 mM Potassium chloride and 20 mM Tris-HCI [pH 8.0].
- Lysis Mix comprised of L1 , L2, L3, dNTPs, PTO-IPC stock:
- Tris-HCI [pH 8.5], 10 mM Potassium chloride and 10 ⁇ EDTA;
- Method 1 The Cognitor Minus test on E. coli spiked blood broth with and without the 95 °C step
- Escherichia coli (ATCC® 25922TM) was grown in nutrient broth for 18 hours at 37°C.
- BacT/ALERT SA blood broth (sheep blood) was inoculated to approximately 1 x 10 7 cfu/mL, 1 x 10 6 cfu/mL, 1 x 10 5 cfu/mL, 1 x 10 4 cfu/mL, and 1 x 10 3 cfu/mL with E. coli.
- Two sets of 1 ml samples were prepared for testing with or without the 95°C step.
- Total viable count (TVC) plates were prepared to confirm cfu/mL values.
- BacT/ALERT blood broth SA was inoculated to approximately 1 x 10 7 cfu/mL and 1 x 10 4 cfu/mL with E. coli.
- Four sets of 1 ml samples were prepared to compare Cognitor Minus test results with and without the use of PTOs, with and without the 95 °C step.
- TVC plates were prepared to confirm cfu/mL values.
- NSCs and positive controls were also prepared (see Table 3). All samples were processed according to the general protocol described above in "Method 1 ".
- samples 9-16 were progressed immediately to QPCR setup, whilst the 95°C (+) samples (samples 1 -8) were incubated at 95°C fa 5 minutes before QPCR setup. Both sets of samples were analysed by QPCR immediately. The same samples were analysed by QPCR after 2 hours at room temperature, and again following a further 18 hours at 4°C.
- NSC no spike control
- UMO unmodified oligonucleotide
- PTO phosphorothioate oligonucleotide
- LM lysis mix
- Figure 1 1 A-B shows the Ct values obtained for E. coli spiked blood broth samples (1 x 10 7 cfu/mL and 1 x 10 4 cfu/mL) and positive controls processed using either UMO LM ( Figure 1 1 A) or PTO LM ( Figure 1 1 B) with and without the 95°C step.
- the data shown here is from a single experiment.
- the Ct values for NSCs using UMO LM were all at least 5 Ct units higher than the Ct values obtained for 1 x 10 3 cfu/mL E. coli spiked blood broth samples, whilst the PTO LM NSC Ct values were all greater than 42.0 Ct units or had no QPCR amplification at all (data not shown).
- substrate primary panel microorganisms
- PTO phosphorothioate modified oligonucleotide
- UMO unmodified oligonucleotide
- the earlier steps in the Cognitor Minus test aim to lyse blood cells and wash away blood- derived proteins such as DNA polymerases, which will produce non-microorganism derived ETGA template DNA, and nuclease enzymes which may digest microorganism- derived ETGA template DNA. This process should not harm any microorganisms that are present in the blood sample. Isolated intact microorganisms are then lysed by the addition of lysis mix (LM) and bead milling. After microorganism lysis and the ETGA reaction, samples contain a mixture of microorganism proteins, LM components, newly synthesised ETGA template DNA and residual blood cell proteins.
- LM lysis mix
- the 95°C step is intended to cenature all proteins in order to protect ETGA template DNA and internal process control (IPC) DNA from nuclease digestion so that it can be successfully detected by QPCR.
- the 95 °C step also inactivates DNA polymerases, thereby quenching the ETGA reaction.
- the UMOs used to formthe ETGA substrate (and IPC) in the LM have been replaced with PTOs which are nuclease resistant.
- the ETGA extension strand that forms the ETGA template is constructed from standard dNTPs, the PTO substrate DNA that it is annealed to may confer protection against nuclease digestion.
- the Cogritor Minus test was carried out using microorganism-spiked blood broth with or without the 95°C step. Samples were analysed by QPCR at five time points: immediately after sample preparation; after 2 hours stored at room temperature (approximately 19 °C); and after 24 hours, 48 hours and 72 hours stored at 4°C.
- Escherichia coli ATCC® 25922TM
- Staphylococcus aureus ATCC® 25923TM
- Candida albicans ATCC® 10231TM
- E. coli and S. aureus in nutrient broth
- C. albicans in Sabouraud media
- BacT/ALERT SA blood broth was inoculated with E. coli, S. aureus and C. albicans to approximately 1 x 10 4 cfu/mL, 1 x 10 4 cfu/mL and 1 x 10 5 cfu/mL respectively.
- Each pellet was resuspended in 500 ⁇ _ Reagent C by tip mixing, transferred to a beadmill tube containing glass beads (0.1 mm and 0.5 mm glass beads), and centrifuged for 3 minutes at 7300 RCF. Following centrifugation, supernatants were transferred to waste by pipette. 50 ⁇ _ LM was added to each sample and an additional 10 ⁇ of DNA Polymerase solution was added to the positive control samples. Samples were then placed into a Disruptor Genie and run for 6 min at 2800 rpm. After bead milling, samples were transferred to a heat block set at 37°C and incubated for 20 minutes.
- PC positive control
- NSC no spike control
- UMO unmodified oligonucleotide
- PTO phosphorothioate oligonucleotide
- LM lysis Table 6 - Materials
- NSC samples processed with PTO LM did not produce Ct values due to low QPCR amplification, and therefore this data is not shown in Figure 12A-E.
- the incidence of sufficient amplification for Ct values was higher for 95 °C (-) samples than 95 °C (+) samples (7/15 Ct valies compared to 2/15 Ct values with no obvious trends for storage duration; and all NSC PTO LM Ct values were between 42.0 Ct units and 45.0 Ct units).
- NSC samples processed with UMO LM produced Ct values at ⁇ hours' that were on average 0.42 Ct units lower without the 95°C step. This increase in QPCR signal for NSC samples processed without the 95°C step is expected given the general increase in signal observed for positive samples.
- the 95 °C step has a significant effect on Ct valuefor all microorganisms and positive controls with PTO LM and UMO LM.
- Sample storage duration (Time' and Time' interactions) has no significant effect on Ct value for any microorganism or positive control with PTO LM; and is also non-significant for C. albicans and the positive control with UMO LM.
- sample storage duration is a significant variable for E. coli and S. aureus with UMO LM: most likely due to the observed reduction in Ct value over time for samples processed without the 95°C step in these sample sets.
- Statistical analysis could not be performed on the NSC PTO LM dataset due to missing Ct values as a result of low QPCR amplification. Summary
- ETGA template DNA detection is improved when the 95 °C step is removed. Furthermore, ETGA QPCR sgnal does not deteriorate with increased sample storage duration in the absence of the 95 °C step when samples are processed using PTO ETGA substrate DNA. ETGA signal is not as stable when samples are processed using UMO ETGA substrate DNA: in the absence of the 95 °C step ETGA
Abstract
Description
Claims
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JP2017521621A JP6768645B2 (en) | 2014-07-10 | 2015-07-10 | Methods and kits for detecting the absence of microorganisms |
CN201580036500.3A CN106661625B (en) | 2014-07-10 | 2015-07-10 | Method and kit for detecting the absence of microorganisms |
EP15738453.8A EP3167077B1 (en) | 2014-07-10 | 2015-07-10 | Method and kit of detecting the absence of micro-organisms |
CA2947801A CA2947801A1 (en) | 2014-07-10 | 2015-07-10 | Method and kit for detecting microorganisms via microbial nucleic acid modifying activity |
ES15738453T ES2698964T3 (en) | 2014-07-10 | 2015-07-10 | Method and detection kit for the absence of microorganisms |
US15/325,225 US10793917B2 (en) | 2014-07-10 | 2015-07-10 | Method and kit of detecting the absence of micro-organisms |
GB1516796.8A GB2542576A (en) | 2014-07-10 | 2015-07-10 | Method and kit of detecting the absence of micro-oranisms |
US17/002,559 US20210130873A1 (en) | 2014-07-10 | 2020-08-25 | Method and Kit of Detecting the Absence of Micro-Organisms |
US17/844,498 US11746389B2 (en) | 2014-07-10 | 2022-06-20 | Method and kit of detecting the absence of micro-organisms |
US18/352,722 US20240018605A1 (en) | 2014-07-10 | 2023-07-14 | Method and Kit of Detecting the Absence of Micro-Organisms |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017182775A1 (en) * | 2016-04-18 | 2017-10-26 | Momentum Bioscience Limited | Microorganism detection involving filtration |
WO2021069903A1 (en) | 2019-10-08 | 2021-04-15 | Momentum Bioscience Limited | Microorganism capture from antimicrobial-containing solution |
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GB201412316D0 (en) * | 2014-07-10 | 2014-08-27 | Momentum Bioscience Ltd | Detecting the absence of micro-organisms |
CN111455021A (en) * | 2019-01-18 | 2020-07-28 | 广州微远基因科技有限公司 | Method and kit for removing host DNA in metagenome |
CN112051398A (en) * | 2020-09-18 | 2020-12-08 | 华侨大学 | Ampicillin and kanamycin joint detection test strip |
CN116694472A (en) * | 2023-07-18 | 2023-09-05 | 墨卓生物科技(浙江)有限公司 | Microorganism lysate, lysis method and kit |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990006320A1 (en) * | 1988-11-30 | 1990-06-14 | Kaellander Clas Fredrik Runess | Substrate for polymerase activity determination |
WO2013103744A1 (en) * | 2012-01-05 | 2013-07-11 | Zeus Scientific, Inc. | Improved dna polymerase activity assays and methods enabling detection of viable microbes |
WO2013155361A1 (en) * | 2012-04-12 | 2013-10-17 | Zeus Scientific, Inc. | Methods for measuring polymerase activity useful for sensitive, quantitative measurements of any polymerase extension activity and for determining the presence of viable cells |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5939262A (en) | 1996-07-03 | 1999-08-17 | Ambion, Inc. | Ribonuclease resistant RNA preparation and utilization |
DE60332468D1 (en) | 2002-08-29 | 2010-06-17 | Ge Healthcare Bio Sciences | analyte |
CA2521520A1 (en) | 2003-04-29 | 2004-11-11 | Amersham Biosciences Corp | Multiply-primed amplification of nucleic acid sequences |
EP1668148B1 (en) | 2003-09-04 | 2008-12-31 | Human Genetic Signatures PTY Ltd. | Nucleic acid detection assay |
EP1711517A4 (en) | 2004-01-21 | 2008-02-13 | Univ Utah Res Found | MUTANT SODIUM CHANNEL NAv1.7 AND METHODS RELATED THERETO |
JP2005269957A (en) | 2004-03-24 | 2005-10-06 | National Institute Of Advanced Industrial & Technology | Method for detecting nucleic acid-binding protein |
GB0703996D0 (en) | 2007-03-01 | 2007-04-11 | Oxitec Ltd | Nucleic acid detection |
ES2644258T5 (en) * | 2010-04-16 | 2021-02-02 | Momentum Bioscience Ltd | Useful Enzyme Activity Measurement Methods for Determining Cell Viability in Non-Purified Samples |
EP3187598B1 (en) | 2011-01-24 | 2018-12-19 | Takara Bio Inc. | Method for modifying nucleic acids |
US8894946B2 (en) | 2011-10-21 | 2014-11-25 | Integenx Inc. | Sample preparation, processing and analysis systems |
CN103436608B (en) * | 2013-08-08 | 2015-02-25 | 中国科学院广州生物医药与健康研究院 | Rapid detection method based on nucleic acid aptamers and kit |
GB201412316D0 (en) * | 2014-07-10 | 2014-08-27 | Momentum Bioscience Ltd | Detecting the absence of micro-organisms |
-
2014
- 2014-07-10 GB GBGB1412316.0A patent/GB201412316D0/en not_active Ceased
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990006320A1 (en) * | 1988-11-30 | 1990-06-14 | Kaellander Clas Fredrik Runess | Substrate for polymerase activity determination |
WO2013103744A1 (en) * | 2012-01-05 | 2013-07-11 | Zeus Scientific, Inc. | Improved dna polymerase activity assays and methods enabling detection of viable microbes |
WO2013155361A1 (en) * | 2012-04-12 | 2013-10-17 | Zeus Scientific, Inc. | Methods for measuring polymerase activity useful for sensitive, quantitative measurements of any polymerase extension activity and for determining the presence of viable cells |
Non-Patent Citations (3)
Title |
---|
D. R. ZWEITZIG ET AL: "Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes", NUCLEIC ACIDS RESEARCH, vol. 40, no. 14, 11 April 2012 (2012-04-11), pages e109 - e109, XP055106744, ISSN: 0305-1048, DOI: 10.1093/nar/gks316 * |
DANIEL R ZWEITZIG ET AL: "Feasibility of a Novel Approach for Rapid Detection of Simulated Bloodstream Infections via Enzymatic Template Generation and Amplification (ETGA)eMediated Measurement of Microbial DNA Polymerase Activity", THE JOURNAL OF MOLECULAR DIAGNOSTICS, 3 May 2013 (2013-05-03), XP055213472 * |
DANIEL R. ZWEITZIG ET AL: "Measurement of Microbial DNA Polymerase Activity Enables Detection and Growth Monitoring of Microbes from Clinical Blood Cultures", PLOS ONE, vol. 8, no. 10, 14 October 2013 (2013-10-14), pages e78488, XP055213350, DOI: 10.1371/journal.pone.0078488 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017182775A1 (en) * | 2016-04-18 | 2017-10-26 | Momentum Bioscience Limited | Microorganism detection involving filtration |
WO2021069903A1 (en) | 2019-10-08 | 2021-04-15 | Momentum Bioscience Limited | Microorganism capture from antimicrobial-containing solution |
EP4283300A2 (en) | 2019-10-08 | 2023-11-29 | Momentum Bioscience Limited | Microorganism capture from antimicrobial-containing solution |
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GB201516796D0 (en) | 2015-11-04 |
ES2698964T3 (en) | 2019-02-06 |
US20220372558A1 (en) | 2022-11-24 |
CN106661625B (en) | 2021-10-26 |
GB201412316D0 (en) | 2014-08-27 |
EP3167077B1 (en) | 2018-10-31 |
GB2542576A (en) | 2017-03-29 |
US10793917B2 (en) | 2020-10-06 |
EP3167077A1 (en) | 2017-05-17 |
US20170240957A1 (en) | 2017-08-24 |
CA2947801A1 (en) | 2016-01-14 |
US20210130873A1 (en) | 2021-05-06 |
JP6768645B2 (en) | 2020-10-14 |
CN106661625A (en) | 2017-05-10 |
US20240018605A1 (en) | 2024-01-18 |
US11746389B2 (en) | 2023-09-05 |
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