WO2011002319A2 - Blocage, extraction et détection combinés d'acide nucléique dans un réceptacle de réaction unique - Google Patents

Blocage, extraction et détection combinés d'acide nucléique dans un réceptacle de réaction unique Download PDF

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WO2011002319A2
WO2011002319A2 PCT/NZ2010/000137 NZ2010000137W WO2011002319A2 WO 2011002319 A2 WO2011002319 A2 WO 2011002319A2 NZ 2010000137 W NZ2010000137 W NZ 2010000137W WO 2011002319 A2 WO2011002319 A2 WO 2011002319A2
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nucleic acid
reagent
amplification
amplifying
sample
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PCT/NZ2010/000137
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WO2011002319A3 (fr
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David James Saul
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Zygem Corporation Limited
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Priority to JP2012518502A priority Critical patent/JP2012531907A/ja
Priority to EP10794424.1A priority patent/EP2449125A4/fr
Publication of WO2011002319A2 publication Critical patent/WO2011002319A2/fr
Publication of WO2011002319A3 publication Critical patent/WO2011002319A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • This application relates generally to a method that can be practiced within a closed system for the rapid amplification of target nucleic acid in a sample. More specifically, the application relates to a combined method for deactivating contaminating nucleic acids and extracting target nucleic acids that is compatible with temperature-controlled target nucleic acid amplification methods which can be used in a closed-system device for the detection of target nucleic acids present in a sample.
  • Processes that accurately and robustly amplify nucleic acids in a sample are desirable for a variety of medical, industrial, environmental, security, research and quality control purposes.
  • such processes are also rapid, yield accurate results and can be performed in a closed-system, i.e. a system that does not need to be opened during the course of the analysis in order to prevent yield reduction or accidental contamination with unwanted nucleic acids or nucleases.
  • Nucleic acid amplification strategies can be split into three stages: deactivation or removal of contaminating nucleic acids, extraction of target nucleic acid, and amplification of target nucleic acid. A primary difficulty with these stages is that they generally require different processes to perform them.
  • Amplification of nucleic acids is very susceptible to contamination.
  • the ability of the polymerase chain reaction (PCR) to amplify minute amounts of DNA is one of its greatest strengths. However, this ability also means that any traces of contaminating DNA are also co- amplified. This amplification of contaminating DNA can produce misleading, ambiguous or incorrect results (Wilson et al., 1990).
  • eubacterial DNA is ubiquitous in the environment and can also contaminate a PCR mix at practically any stage of processing by laboratory personnel (Kitchin et al., 1990; Millar et al., 2002).
  • Other sources of DNA contamination include: aerosols (Saksena et al., 1991), consumable PCR reagents, plastic ware (Millar et al., 2002), and commercial PCR primers (Goto et al, 2005).
  • mtDNA mitochondrial DNA
  • Sequence analysis of human mtDNA is widely used for forensic purposes to characterize specimens when there is insufficient nuclear DNA. Often, mtDNA is the only remaining intact DNA in trace or degraded samples. Handling these types of samples requires rigorous quality assurance because the laboratories in which they are processed are often a more abundant source of DNA than the sample itself. (Ca ⁇ racedo et al., 2000).
  • amplification rely on a pre-treatment step either with nucleic acid degrading enzymes or with chemical agents that inactivate the nucleic acid contaminants.
  • Example enzymes used in the treatment of reagents are DNAseI or SauSA. Solutions for RNA analysis are often treated with diethyl pyrocarbonate (DEPC) and double-stranded DNA can be removed using ethidium monoazide (New Zealand Patent No. 545,894).
  • DEPC diethyl pyrocarbonate
  • ethidium monoazide New Zealand Patent No. 545,894
  • nucleic acid extraction techniques are problematic as the sample tube may require opening and shutting at stages throughout the extraction procedure. Contamination may occur simply as a result of the sample tube being opened to the atmosphere or being touched by a technician.
  • a target nucleic acid can be subjected to nucleic acid amplification methods to determine if the target nucleic acid is present in a sample.
  • nucleic acid amplification methods Many situations arise where it is desirable to detect low levels of specific nucleic acid sequences within the context of a complex mixture, therefore any of the current methods for detecting nucleic acids chosen for this purpose must be highly specific and sensitive.
  • the most widespread method used to achieve this goal is PCR. This method provides exponential amplification of target molecules by using thermal cycling and a thermostable DNA polymerase.
  • the methods, processes, and devices disclosed herein overcome the shortcomings and disadvantages mentioned above.
  • methods for combining deactivation of contaminating nucleic acids, extraction of target nucleic acid, and amplification of target nucleic acid into a single process that can be practiced in a closed-system are described.
  • the methods of deactivation of contaminating nucleic acids disclosed herein can be controlled through application of an external stimulus, and surprisingly are compatible with most strategies of nucleic acid extraction and amplification.
  • the conditions required for nucleic acid extraction by thermophilic proteinase treatment are compatible with those of most amplification processes.
  • One embodiment provides a method of decontaminating, extracting and amplifying target nucleic acid in a closed system comprising a single vessel or tube, the method including: i) adding nucleic acid deactivating reagent that has an active form and an inactive form, is converted to the active form only when an external stimulus is applied, and only the active form deactivates contaminating nucleic acids in the system causing contaminating nucleic acids to become unreactive to nucleic acid amplifying reagent; thermophilic proteinase that is in an active form at about 65- 8O 0 C and in an inactive form at or above about 90 0 C; and nucleic acid amplifying reagent to a sample comprising target nucleic acid to form the system, ii) closing the system;
  • nucleic acid deactivating reagent from the inactive form to the active form to cause contaminating nucleic acids in the system to become unreactive to nucleic acid amplifying reagent and subsequently having the nucleic acid deactivating reagent be converted to the inactive from;
  • thermophilic proteinase and extract the target nucleic acid for amplification by effecting one or more of lysis of cells, digestion of proteins, and digestion of cell- wall enzymes; v) incubating the sample at or above about 9O 0 C to cause auto-catalysis of the thermophilic proteinase; and
  • the nucleic acid deactivating reagent is converted to the inactive form before or concurrent with incubating the sample at about 65-8O 0 C.
  • the nucleic acid deactivating reagent is converted to the inactive form subsequent to causing contaminating nucleic acids to become unreactive to nucleic acid amplifying reagent by applying the external stimulus for a period of time sufficient to convert the nucleic acid deactivating reagent to the inactive form.
  • the nucleic acid deactivating reagent is converted to the inactive form subsequent to causing contaminating nucleic acids to become unreactive to nucleic acid amplifying reagent by removing the external stimulus.
  • the nucleic acid deactivating reagent is provided at a concentration sufficient to deactivate contaminating nucleic acids without inhibiting the thermophilic proteinase, the amplifying reagent, or amplification of the target nucleic acid.
  • the nucleic acid deactivating reagent is a mesophilic nucleic acid modifying enzyme.
  • the mesophilic nucleic acid modifying enzyme is a double-stranded specific endonuclease, a double-stranded specific exonuclease, a single-stranded specific nuclease, a restriction endonuclease, an RNAses, RNAseH, or an RNA modifying enzyme.
  • the mesophilic nucleic acid modifying enzyme is activated by incubating at about 25-40 0 C for a period of time sufficient to convert the mesophilic nucleic acid modifying enzyme to the active form.
  • the mesophilic nucleic acid modifying enzyme is inactivated by incubating at about 65-8O 0 C for a period of time sufficient to convert the mesophilic nucleic acid modifying enzyme to the inactive form.
  • the nucleic acid deactivating reagent is a nucleic acid intercalating agent.
  • applying the external stimulus converts the nucleic acid intercalating agent to the active form by effecting photolysis of the nucleic acid intercalating agent.
  • the active form of the nucleic acid intercalating agent covalently binds to double-stranded nucleic acids.
  • the nucleic acid intercalating agent is ethidium monoazide.
  • the ethidium monoazide is provided at a concentration between about 1 ⁇ g/ml and about 5 ⁇ g/ml.
  • the ethidium monoazide is provided at a concentration of about 3 ⁇ g/ml.
  • the external stimulus is a specific narrow spectrum wavelength light, broad spectrum white light, or UV light.
  • the external stimulus is specific narrow spectrum wavelength light, broad spectrum white light, or UV light sufficient to activate the nucleic acid intercalating agent by effecting photolysis of the nucleic acid intercalating agent.
  • the external stimulus is thermal energy sufficient to activate the nucleic acid deactivating agent without substantially activating the thermophilic proteinase.
  • the external stimulus is thermal energy sufficient to raise the system to a temperature of about 25-4O 0 C for a period of time sufficient to convert the mesophilic nucleic acid modifying enzyme to the active form.
  • thermophilic proteinase is EAl.
  • the method further includes:
  • the mesophilic enzyme is a cellulase or lysozyme.
  • amplifying is detected by fluorescence.
  • amplifying comprises performing a PCR detection method.
  • the PCR detection method is qPCR, multiplex PCR, or reverse-transcription PCR.
  • the amplifying comprises performing an isothermal detection method.
  • the isothermal detection method is strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification, isothermal chimeric primer-initiated amplification of nucleic acids, Q-beta amplification systems, or OneCutEventAmplificatioN.
  • the isothermal detection method utilizes a technique such as Nuclease Chain Reaction (NCR), RNAse-mediated
  • RNCR Nucleases Chain Reaction
  • PNCR Polymerase Nuclease Chain Reaction
  • RMD RNAse- Mediated Detection
  • TR-REF Tandem Repeat Restriction Enzyme Facilitated Chain Reaction
  • IRC-REF Inverted reverse Complement Restriction Enzyme Facilitated
  • amplifying comprises performing a SNP detecting assay.
  • amplifying the target nucleic acid is automated.
  • the amplifying reagent is added by microfluidics or a solid dispenser.
  • the amplifying reagent is added by microcapsules comprising the amplifying reagent.
  • the microcapsules are predisposed in the vessel or tube.
  • the microcapsules are heat-labile capsules.
  • the heat-labile capsules are agarose or wax beads.
  • the heat-labile capsules release detecting reagents when exposed at a sufficient temperature to melt or dissolve the capsules.
  • the amplifying reagent is resistant to proteolytic cleavage by the thermophilic proteinase.
  • the sample is blood, urine, saliva, semen, stool, tissue, swabs, tears, bone, teeth, hair, or mucus.
  • the sample is bacteria, fungi, archaea, eukarya, protozoa, or virus.
  • the sample is a mixture of salt water, freshwater, ice, soil, waste material, or food.
  • the vessel or tube is a device.
  • the device is a hand-held device.
  • the device or components of the device are disposable.
  • the device comprises an inlet port, an outlet port, a chamber, a detector for emitted fluorescence and an excitation light source.
  • the device further comprises microfluidics, microchips, nanopore technologies and miniature devices.
  • the device comprises a sealed unit, a light-tight chamber, a nucleic acid deactivating reagent-activating light source, a detector for emitted fluorescence and an excitation light source.
  • the nucleic acid deactivating reagent- activating light source is an incandescent lamp, a fluorescent lamp, or an array of light emitting diodes.
  • Another embodiment provides a method of amplifying target nucleic acid in a closed system comprising a single vessel or tube, the method including:
  • mesophilic nucleic acid modifying enzyme that has an active form and an inactive form where only the active form deactivates contaminating nucleic acids in the system causing the contaminating nucleic acids to become unreactive to PCR amplifying reagent and is in an active from at about 25- 4O 0 C and in an inactive form at about 65-8O 0 C; EAl proteinase that is in an active from at about 65-8O 0 C and is in an inactive form at or above about 90°C; and PCR amplifying reagent to a sample comprising target nucleic acid to form the system,
  • Yet another embodiment provides a method of amplifying target nucleic acid in a closed system comprising a single vessel or tube, the method including:
  • ethidium monoazide that has an active form and an inactive form and only the active form deactivates contaminating nucleic acids in the system causing the contaminating nucleic acids to become unreactive to PCR amplifying reagent, and is converted to the active form only when specific narrow spectrum wavelength light, broad spectrum white light, or UV light is applied to effect photolysis of the ethidium monoazide; EAl proteinase that is in an active form at about 65-8O 0 C and is in an inactive from at or above about 90°C; and PCR amplifying reagent to a sample comprising target nucleic acid to form the system,
  • FIGURES BRIEF DESCRIPTION OF THE FIGURES [0034]
  • Figure 1 Overview of combined nucleic acid detection process.
  • Figure 2 Single chamber device with an array of light emitting diodes for deactivation of contaminating nucleic acids, extraction of target nucleic acid, and amplification of target nucleic acid using encapsulated reagents.
  • FIG. 3 A graph showing the C T values obtained in a qPCR reaction for different Escherichia coli cell counts when DNA extraction and qPCR are performed in a single vessel. In this reaction, no ethidium monoazide treatment was used. The dashed line indicates the C T value obtained from water alone (zero cells). Error bars are one standard deviation from the mean.
  • FIG. 4 A graph showing the C T values obtained in a qPCR reaction for different Escherichia coli cell counts when DNA extraction and qPCR are performed in a single vessel. In this reaction, an ethidium monoazide treatment is included as part of a closed-tube sequential reaction. The dashed line indicates the C T value obtained from water alone (zero cells). Error bars are one standard deviation from the mean.
  • nucleic acid deactivating reagent refers to any chemical, molecule, enzyme, or other type of biological molecule capable of making nucleic acids unreactive to nucleic acid amplification.
  • deactivation refers to the process of removing, degrading, or making contaminating nucleic acids unreactive to nucleic acid amplification.
  • the term "unreactive" refers to a nucleic acid being unable to be amplified by any nucleic acid amplification step or method.
  • a non-limiting example is ethidium monoazide, which can bind the contaminating nucleic acid, which remains in the solution but is not detectable by the signal detection methods used subsequent to target nucleic acid
  • Target nucleic acid amplification strategies can be split into three stages: deactivation of contaminating nucleic acids, extraction of target nucleic acid, and amplification of target nucleic acid. Any system for performing these stages requires different instrumentation for each stage, and a method of transferring the material from one internal instrument to the next.
  • the art teaches discrete steps for nucleic acid pre-treatment, extraction and amplification and although each of these may be in closed systems, those systems must be opened and exposed to potential contamination between each step.
  • the methods for deactivating contaminating nucleic acids disclosed herein can be controlled through application of an external stimulus, and surprisingly are compatible and can be successfully included in a single closed system with nucleic acid extraction and amplification methods.
  • sample comprising target nucleic acid, nucleic acid deactivating reagent, nucleic acid extraction reagent, and nucleic acid amplification reagent, are added to an open vessel or tube and the vessel or tube is sealed to prevent further contamination.
  • the vessel or tube is opened for a period of time sufficient to introduce additional reagents, such as nucleic acid extraction reagent or nucleic acid amplification reagent.
  • a system is open when the vessel or tube is opened after nucleic acid deactivation, before or after extraction, or before amplification.
  • inclusion of a nucleic acid deactivation reagent improves the amplification detection sensitivity of a method by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5 -fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 1000-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, at least about 1, 000-fold, at least about 2,000-fold, at least about 3,000-fold, at least about 4,000-fold, at least about 5,000-fold, or greater than amplification detection sensitivity of the same method performed without the nucleic acid deactivating reagent.
  • amplification detection sensitivity of a method practiced in a closed system including nucleic acid deactivation reagent increases by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 1000-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, at least about 1, 000-fold , at least about 2,000-fold, at least about 3,000-fold, or greater compared to the same method performed in an open system.
  • thermophilic proteinase treatment is temperature controlled, as are all amplification methods, hi addition, the conditions required for the thermophilic treatment are compatible with those of most amplification processes. Because of these factors, combined methods for deactivating contaminating nucleic acids, extracting a target nucleic acid, and amplifying the target nucleic acid can be performed in a single vessel or device. Furthermore, the device can be simplified to a vessel with an external stimulus source to activate a nucleic acid deactivating reagent for deactivation of contaminating nucleic acids, and a heating/cooling mechanism to process raw sample material to take it all the way to amplification of a detectable target nucleic acid. The inclusion of a detector is also facile.
  • the currently disclosed methods allow for combined processes for deactivation of contaminating nucleic acids, extraction of target nucleic acid, and amplification of target nucleic acid, and allow for devices with no pumps or need for microfluidics, however these can be used for more complex downstream applications if required. Furthermore, the combined process can be practiced in closed-system devices that utilize external stimuli and heat-controlled reaction chains. Nucleic acid deactivating reagents and thermophilic proteinases are used to inhibit contaminating nucleic acids in the system and to extract nucleic acids in a sample in tandem with nucleic acid amplification techniques including PCR or isothermal amplification.
  • Nucleic acid deactivating reagents that can be activated by application of an external stimulus and can be subsequently deactivated, as well as heat control using either temperature dependent enzyme mixtures or temperature controlled release of encapsulated reagents simplify the design of current nucleic acid diagnostic methods and devices.
  • the inclusion of a deactivation step as part of the process reduces the rate of false positives and increases the sensitivity of nucleic acid amplification and detection. Reducing complexity can also reduce associated failure rate and cost.
  • nucleic acid deactivating reagent in a first step nucleic acid deactivating reagent, thermophilic proteinase, nucleic acid amplification and detection reagents, and sample are added to a single reaction vessel.
  • An external stimulus can then be applied to convert a nucleic acid deactivating reagent from an inactive form to an active form.
  • the active form of the nucleic acid deactivating reagent then reacts with all contaminating nucleic acids and renders them unreactive to all subsequent steps in the process.
  • the unused nucleic acid deactivating reagent is converted to an inactive form.
  • thermophilic proteinase then digests contaminating proteins at a temperature optimal for thermophilic proteinase activity and effects the extraction of target nucleic acid. Subsequent to extraction of target nucleic acid, known nucleic acid sequences can be amplified by PCR or isothermal methods, or simultaneously detected by fluorescence using isothermal amplification or qPCR amplification methods.
  • nucleic acid deactivating contaminating nucleic acids Preferred methods for deactivating contaminating nucleic acids and preventing co- amplification of the contaminating nucleic acids by using a nucleic acid deactivating reagent are described herein.
  • nucleic acid sequence As used herein, the terms "nucleic acid”, “nucleic acid sequence”,
  • polynucleotide(s),” “polynucleotide sequence” and equivalents thereof mean a single or double- stranded deoxyribonucleotide or ribonucleotide polymer of any length, and include as non- limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polynucleotides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers, fragments, genetic constructs, vectors and modified polynucleotides.
  • nucleic acid nucleic acid
  • oligonucleotide and polynucleotide
  • the nucleic acid deactivating reagent be used in combination with the nucleic acid extraction and amplification reactions requiring the least possible number of opening, adding or removals from the reaction tube.
  • the reagent is added in one initial step with any nucleic acid extraction and amplification reagents required for amplification and detection of a target nucleic acid.
  • the nucleic acid deactivating reagent can be a chemical, an enzyme, or other type of biological molecule. Any type of nucleic acid deactivating reagent will have the ability to deactivate contaminating nucleic acids.
  • Contaminating nucleic acid is. any nucleic acid that may be found in nucleic acid preparation/extraction reagents; nucleic acid amplification reagents; in vessels, tubes, plates, or devices used for any process involving nucleic acids; or plastic-ware used in any process involving nucleic acids that contaminate the target sample and lead to a reduction in amplification sensitivity and to an increase in the number of false positive results.
  • the nucleic acid deactivating reagent has properties which allow it to remain in the sample solution during target nucleic acid extraction and amplification. This is advantageous in that it reduces the possibility for further contamination by opening the container/reaction vessel and allows deactivation to be incorporated into a nucleic acid amplification process.
  • the nucleic acid deactivating reagent has two different forms under conditions compatible with the methods described herein ⁇ an inactive form where it deactivates nucleic acids, and an active form where it deactivates nucleic acids, and the
  • nucleic acid deactivating reagent may alternate between an inactive and an active form (and vice versa), such that a sufficient quantity of the nucleic acid deactivating reagent to deactivate contaminating nucleic acids can be added to the sample and will only deactivate the contaminating nucleic acids when in an active form.
  • any remaining nucleic acid deactivating reagent can be converted to an inactive form that will not deactivate any further nucleic acids. This prevents the nucleic acid deactivating reagent/contaminating nucleic acid combination from interfering in any further reactions in the target nucleic acid amplification process described herein.
  • the nucleic acid deactivating reagent is converted to an active form prior to target nucleic acid extraction and amplification.
  • the nucleic acid deactivating reagent is converted to an inactive form subsequent to causing contaminating nucleic acids to become unreactive to nucleic acid amplification and prior to or concurrent with incubating a sample with nucleic acid extraction reagent such as a thermophilic proteinase. Conversion to or from the active form may occur via any of several mechanisms depending on the properties of the particular nucleic acid deactivating reagent. Exemplary mechanisms are discussed further below.
  • the nucleic acid deactivating reagent is provided in active form at a concentration sufficient to deactivate contaminating nucleic acids without inhibiting subsequent target nucleic acid extraction including treating a sample with thermophilic proteinase, nucleic acid amplification reagent, or target nucleic acid amplification.
  • the nucleic acid deactivating reagent does not break down or destroy the contaminating nucleic acids, as such nucleic acid fragments or portions may themselves interfere in the amplification reaction.
  • the nucleic acid deactivating reagent will only be converted to an active form when an external stimulus is applied.
  • the external stimulus is sufficient to activate the nucleic acid deactivating reagent without substantially activating a nucleic acid extraction reagent such as a thermophilic proteinase.
  • substantially activating refers to, for example, a thermophilic proteinase becoming sufficiently active to effect the extraction of an amount of target nucleic acid sufficient to be amplified and detected by the methods described herein during a period of time sufficient to convert the nucleic acid deactivating reagent to an active form.
  • the activating external stimulus is specific narrow spectrum wavelength light, broad spectrum white light, or UV light, while in other preferred embodiments the external stimulus is thermal energy.
  • the external stimulus will depend on the nucleic acid deactivating reagent selected and its specific activating and deactivating properties.
  • the nucleic acid deactivating reagent subsequent to causing contaminating nucleic acids to become unreactive to nucleic acid amplification, is converted to its inactive form.
  • removal of the external stimulus converts the active nucleic acid deactivating reagent to an inactive form.
  • a particular amount of external stimulus may be sufficient to convert the nucleic acid deactivating reagent to an active form, and an increase in the amount of the external stimulus may be required to convert the active nucleic acid deactivating reagent to an inactive form.
  • the nucleic acid deactivating reagent is converted to an inactive form subsequent to causing contaminating nucleic acids to become unreactive to nucleic acid amplification by applying an external stimulus for a period of time sufficient to convert the nucleic acid deactivating reagent to an inactive form.
  • the external stimulus converts the nucleic acid deactivating reagent into an active form by effecting a nonreversible structural change in the nucleic acid deactivating reagent, for example photolysis, that allows for the deactivation of contaminating nucleic acids at the time of activation, but does not allow for the further deactivation of nucleic acids after the structural change has occurred.
  • applying the external stimulus may convert a nucleic acid deactivating reagent to the active form by effecting photolysis of the nucleic acid deactivating reagent.
  • the nucleic acid deactivating reagent is an intercalating agent such as ethidium, proflavin, or thalidomide which covalently binds to double-stranded nucleic acids.
  • intercalating agent includes any agent that can be reversibly inserted between two adjacent base pairs of a double-stranded nucleic acid.
  • Non-limiting examples of well studied DNA intercalators include ethidium, proflavin, and thalidomide.
  • applying specific narrow spectrum wavelength light, broad spectrum white light, or UV light converts a nucleic acid intercalating agent to an active form by effecting photolysis of the nucleic acid intercalating agent.
  • the intercalating agent is ethidium monoazide (EMA).
  • EMA ethidium monoazide
  • New Zealand Patent No. 545,894 which is hereby incorporated by reference, discloses using EMA to deactivate contaminating nucleic acids.
  • EMA is a photoreactive analogue of ethidium bromide in which the amino group at the 8- position has been replaced with an azido group. In the absence of white light, EMA intercalates with double-stranded nucleic acids in the same manner as ethidium bromide.
  • EMA becomes photo-activated and covalently binds double-stranded nucleic acids (Bolton and Kearns, 1978; Garland et. al., 1980).
  • the azido-group at the 8-position of EMA is photo-chemically lysed using long wavelength light greater than approximately 400 nm (Bolton and Kearns, 1978).
  • EMA that has undergone photolysis can covalently crosslink with nucleic acids at the site of binding via a nitrene radical (Hardwick et al., 1984; Cantrell et al., 1979).
  • EMA loses its ability to covalently bind any further unbound nucleic acids. Therefore subsequent exposure to light (e.g. in a real-time PCR machine) does not affect the outcome of any downstream reaction. Furthermore, covalently cross-linked nucleic acids are unable to participate in any subsequent nucleic acid amplification step, and therefore its interference is removed (Nogva et al., 2003).
  • EMA is added at a minimal titer, just sufficient to bind all exogenous nucleic acids, while minimizing the formation of free nitrene in solution associated with the formation of hydroxylamine.
  • the amount of contaminating nucleic acids in commercially available amplification reagents may vary, thus every batch of amplification reagents will require optimization of the EMA titer to be undertaken to ensure maximal removal of contaminating nucleic acids while maintaining amplification sensitivity.
  • the amount of EMA added is sufficient to bind
  • EMA may be used at a concentration of between l ⁇ g/ml and 5 ⁇ g/ml. More preferably, the EMA concentration is about 3 ⁇ g/ml.
  • nucleic acid deactivating reagents may be used.
  • the nucleic acid deactivating reagent should be incapable of diffusing into the cellular material.
  • larger molecules such as a mesophilic nucleic acid modifying enzymes may be used.
  • the enzymes used will not hydrolyse or modify the oligonucleotides necessary for PCR, for example, nucleases that modify or hydrolyse short single-stranded nucleic acids cannot be used.
  • nucleases that modify or hydrolyse short single- stranded nucleic acids may be used if the oligonucleotides used for the amplification step are modified such that they are resistant to cleavage by the nuclease.
  • a 5 1 modification of a single stranded oligonucleotide may be used to render the oligonucleotide resistant to 5 1 - specific exonucleases.
  • the nucleic acid modifying enzyme is combined with a nucleic acid extraction reagent, e.g., thermophilic proteinase, that possesses a different temperature activation profile than the mesophilic nucleic acid modifying enzyme.
  • a nucleic acid extraction reagent e.g., thermophilic proteinase
  • activation of the mesophilic nucleic acid modifying enzyme would involve applying thermal energy to raise the temperature of the sample containing the mesophilic nucleic acid modifying enzyme to a level suitable for activation but not high enough to activate the thermophilic proteinase to any significant level.
  • the mesophilic nucleic acid modifying enzyme may be incubated at about 20 to 45°C for a period of time sufficient to convert the mesophilic nucleic acid modifying enzyme from an inactive from to an active form.
  • the mesophilic nucleic acid modifying enzyme is incubated at about 65 to 80 0 C for a period of time sufficient to convert the mesophilic nucleic acid modifying enzyme from the active form to the inactive form.
  • a mesophilic nucleic acid modifying enzyme such as a nuclease may be used, particularly in combination with PCR, qPCR or RT-PCR amplification methods.
  • the mesophilic nucleic acid modifying enzyme may be a double-strand specific endonuclease (e.g. a restriction endonuclease); a double-strand specific exonuclease; a single-strand specific nuclease with low activity on short molecules; or a nucleic acid modifying enzyme that is specific to double-stranded nucleic acids.
  • a double-strand specific endonuclease e.g. a restriction endonuclease
  • a double-strand specific exonuclease e.g. a restriction endonuclease
  • a single-strand specific nuclease with low activity on short molecules e.g. a restriction endonuclease
  • a nucleic acid modifying enzyme that is specific to double-stranded nucleic acids.
  • the mesophilic nucleic acid modifying enzyme may be T7 exonuclease, T7 endonuclease 1, Exo III, a frequent-cutter restriction endonuclease, or any mixture thereof.
  • mesophilic nucleic acid modifying enzymes may be used to deactivate contaminating nucleic acids associated with reverse transcription PCR.
  • the mesophilic nucleic acid modifying enryme may be an RNAse, an RNAseH, or an RNA modifying enzyme.
  • target nucleic acid can be extracted from the sample.
  • Methods for the treatment of sample with a thermophilic proteinase to extract target nucleic acids are described below.
  • nucleic acid Preferred methods for extracting target nucleic acid from a biological sample comprising cells are described herein.
  • extracting and extraction refer to the process of increasing the availability of nucleic acid within a sample for processing by other manipulations. Implicit in the concept of nucleic acid extraction is that the target nucleic acid is sufficiently free of interfering substances such as inhibitors, nucleases, other enzymes, and nucleoproteins, and that it is effective in other manipulation methods. It is understood that the nucleic acid is not necessarily purified away from non-interfering compounds as to do so serves no purpose in the present device. The nucleic acid treatment minimizes the negative effects of interfering compounds.
  • nucleic acid extraction be performed in combination with amplification reactions requiring the least possible number of opening, adding, or removals from the reaction tube.
  • the extraction occurs in the same reaction vessel as target nucleic acid amplification.
  • US Patent No. 7,547,510 which is hereby incorporated by reference, discloses a method of nucleic acid extraction using thermophilic proteinases that are able to be combined with nucleic acid amplification in a single reaction vessel.
  • Samples can be obtained from a wide range of substrates including clinical, food and beverage or environmental samples. Typically, microbial samples are obtained from
  • tissue samples may be obtained using standard techniques such as cell scrapings or biopsy techniques to collect animal tissue.
  • blood sampling is routinely performed, for example for pathogen testing, and methods for taking blood samples are well known in the art.
  • methods for storing and processing biological samples are well known in the art. For example, tissue samples may be frozen until tested.
  • one of skill in the art would realize that some test samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.
  • Samples for forensic purposes are typically, blood, saliva, semen, skin, hair, bones or teeth.
  • the more problematic samples where the target nucleic acid is considered to be trace or "low copy number" are degraded stains or bones, teeth, and hair from historic cases.
  • Samples used for species identification, anthropology, phylogenetics, and "bar coding" come from a wide variety of sources but include archaeological digs, bones, feathers and hair, and also preserved museum samples such as those stored in formalin, alcohol, or paraffin wax.
  • samples comprising cells from which target nucleic acids are extracted are blood, urine, saliva, semen, stool, tissue, swabs, tears, or mucus samples.
  • the sample is from bacteria, fungi, archaea, eukarya, protozoa, or virus.
  • the sample is a mixture of, salt water, fresh water, ice, soil, waster material, and food.
  • the sample is blood, urine, saliva, semen, stool, tissue, swabs, tears, bone, teeth, hair, or mucus.
  • the sample can also be skin, a feather, or preserved or historic specimens.
  • thermophilic proteinases are used to extract nucleic acid from biological samples.
  • Thermophilic proteinases have protein degradation activity at high temperatures.
  • thermophilic proteinase refers to a proteinase having optimal proteolytic activity at elevated temperatures, for example, from about 65-80 0 C, and includes proteinases isolated from thermophilic organisms and proteinases isolated from organisms other than thermophilic organisms that have optimal proteolytic activity as described above, including any variants of such proteases such as those that have subsequently been modified or engineered.
  • thermophilic proteinases particularly suitable for use in the methods described herein include those that are heat denaturable and/or exhibit auto-catalytic activity at or above about 9O 0 C, and are preferably permanently heat-inactivated at elevated temperatures, for example, at temperatures at or above about 90 0 C.
  • the preferred characteristics for a thermophilic proteinase to be used include: 1) being substantially stable and active within the range of about 65-80°C, and
  • thermophilic proteinase A preferred incubation temperature required to activate the thermophilic proteinase and extract the target nucleic acid by effecting one or more of the lysis of cells, digestion of proteins, digestion of cell-wall enzymes may be 75 0 C.
  • the preferred incubation temperature required to cause auto-catalysis of the thermophilic proteinase may be 95°C.
  • these temperatures are given by way of example only and are not meant to be limiting in any way. It is anticipated that the proteinases will have differing profiles for both enzyme activity and stability over a range of temperatures and that such enzyme dynamics would be known to a skilled artisan. It is also anticipated such enzyme profiles for the proteinases could be determined with minimal experimentation.
  • thermophilic proteinase is EAl proteinase, a neutral proteinase isolated from Bacillus species strain EAl, and variants thereof; and AkI proteinase, a serine protease isolated from Bacillus species strain AkI, and variants thereof.
  • EAl proteinase may be used.
  • EAl proteinase can be activated and degraded (through auto-lysis) via temperature shifts.
  • EAl proteinase is active when incubated at about 65-80 0 C. At this temperature, cells of a sample are lysed and the EAl proteinase degrades contaminating protein and also rapidly removes DNA- degrading nucleases at temperatures where these nucleases are inactive, thereby minimising degradation of the target nucleic acid.
  • extraction of target nucleic acid from a sample in a closed- system includes the steps of:
  • thermophilic proteinase 1) adding at least one thermophilic proteinase to a sample containing nucleic acid for
  • thermophilic proteinase is stable and active at about 65-8O 0 C but is inactivated and/or denatured when the sample is incubated at or above about 9O 0 C without requiring the addition of further denaturing agents.
  • thermophilic proteinase While in preferred embodiments a thermophilic proteinase may be used, this should not been seen as a limitation for other enzymes that could also conceivably be used with the methods described herein.
  • mesophilic enzymes active at lower temperatures than thermophilic proteinases, may be mixed with a thermophilic proteinase and used to weaken and/or remove cell walls from plant, fungal tissue, bacteria, spores and biofilms before activating the thermophilic proteinase and continuing with the closed-system nucleic acid extraction.
  • the practice of the disclosed method relies on the proteinase and/or a proteinase/cell-wall degrading enzyme having differential activities at different temperatures. By cycling through the variable temperatures, the activities of different enzymes can be brought into play without the need for opening the system to add new reagents.
  • a method using mesophilic enzymes with a thermophilic proteinase further includes the steps of: a. adding at least one mesophilic enzyme and at least one non-specific thermophilic enzyme to a sample comprising a target nucleic acid for testing, and b. incubating the sample for a preferred period of time below about 40 0 C as required to effect removal of any cell walls from cells of a sample.
  • a preferred initial incubation temperature required to effect removal of any cell walls via activity of the mesophilic enzyme may be 37°C.
  • the mesophilic enzyme may be a cellulase or a lysozyme.
  • thermophilic proteinases can also be used in combination with other hydrolases that will burst or weaken cell walls. This allows for the extraction of target nucleic acids from recalcitrant cells, such as plant cells, tough bacterial cells, or fungal spores obtained from the environment (such as soil, rock, water and plant material samples, for example) or from subjects, including tissues or fluids from a subject.
  • target nucleic acids such as plant cells, tough bacterial cells, or fungal spores obtained from the environment (such as soil, rock, water and plant material samples, for example) or from subjects, including tissues or fluids from a subject.
  • the target nucleic acid in the sample can then be amplified.
  • Known nucleic acid sequences of interest can be amplified by PCR-based methods or isothermal-based methods described below.
  • the amplification methods may include PCR-based and isothermal-based methods for amplifying target nucleic acids of interest from a sample after the sample has been treated with a nucleic acid deactivating reagent to inhibit any contaminating nucleic acids and the target nucleic acid has been extracted from the sample by any of the methods detailed above.
  • the process of target nucleic acid amplification may be automated.
  • Reagents for PCR typically include a set of primers for each target nucleic acid, a DNA polymerase (preferably a thermostable DNA polymerase), a DNA polymerase cofactor, and one or more deoxyribonucleoside-5' -triphosphates (dNTP's) or similar nucleosides.
  • a DNA polymerase preferably a thermostable DNA polymerase
  • a DNA polymerase cofactor preferably a DNA polymerase cofactor
  • dNTP's deoxyribonucleoside-5' -triphosphates
  • a DNA polymerase is an enzyme that will add deoxynucleoside monophosphate molecules to (usually the 3 '-hydroxy) end of the primer in a complex of primer and template, but this addition is in a template dependent manner. Generally, synthesis of extension products proceeds in the 5' to 3' direction of the newly synthesized strand until synthesis is terminated.
  • Useful DNA polymerases include, for example, Taq polymerase, E. coli DNA polymerase I, T4 DNA polymerase, Klenow polymerase, reverse transcriptase and others known in the art.
  • the DNA polymerase is thermostable meaning that it is stable to heat and
  • thermostable DNA polymerases are not substantially inactive at the high temperatures used in polymerase chain reactions as described herein. Such temperatures will vary depending on a number of reaction conditions, including pH, nucleotide composition, length of primers, salt concentration and other conditions known in the art.
  • Particularly useful polymerases are those obtained from various Thermus bacterial species, such as Thermus aquaticus, Thermus thermophilus, Thermus filiformis, and Thermus flavus.
  • Other useful thermostable polymerases are obtained from various microbial sources including Thermococcus literalis, Pyrococcus furiosus, Thermotoga sp. And those described in WO-A-89/06691 (published JuI. 27, 1989).
  • Some useful thermostable polymerases are commercially available, such as, AmpliTaq ", Tth, and UlTma " from Perkin Elmer, Pfu from Stratagene, and Vent and Deep- Vent from New England Biolabs.
  • Other polymerases are complexed with other molecules that render them inactive until a high temperature is applied. Typically, an antibody is used.
  • An example of such a polymerase is Platinum ® Taq from
  • polymerases from organisms and for producing genetically engineered enzymes using recombinant techniques.
  • a DNA polymerase cofactor refers to a non-protein compound on which the enzyme depends for activity. Thus, the enzyme is catalytically inactive without the presence of cofactor.
  • a number of materials are known co factors including, but not limited to, manganese and magnesium salts, such as chlorides, sulfates, acetates and fatty acids salts. Magnesium chlorides and sulfates are preferred.
  • deoxyribonucleoside-5 '-triphosphates such as two or more of dATP, dCTP, dGTP and dTTP.
  • Analogues such as dITP, dUTP, and 7-deaza- dGTP are also useful.
  • the four common triphosphates dATP, dCTP, dGTP and dTTP are used together.
  • the PCR reagents described herein are provided and used in PCR in suitable concentrations to provide amplification of the target nucleic acid.
  • the minimal amounts of primers, DNA polymerase, cofactors and deoxyribonucleoside-5 '-triphosphates needed for amplification and suitable ranges of each are well known in the art.
  • the minimal amount of DNA polymerase is generally at least about 0.5 units/1 OO ⁇ l of solution, with from about 2 to about 25 units/1 OO ⁇ l of solution being preferred, and from about 7 to about 20 units/1 OO ⁇ l of solution being more preferred. Other amounts may be useful for given amplification systems.
  • a "unit” is defined herein as the amount of enzyme activity required to incorporate 10 nmoles of total nucleotides (dNTP's) into an extending nucleic acid chain in 30 minutes at 74°C.
  • the minimal amount of primer is at least about 0.075 ⁇ Mol with from about 0.1 to about 2 ⁇ Mol being preferred, but other amounts are well known in the art.
  • the cofactor is generally present in an amount of from about 2 to about 15mMol.
  • the amount of each dNTP is generally from about 0.25 to about 3.5mMol.
  • the PCR reagents can be supplied individually, or in various combinations, or all in a buffered solution having a pH in the range of from about 7 to about 9, using any suitable buffer, many of which are known in the art. .
  • thermostable DNA polymerase examples include antibodies specific for the thermostable DNA polymerase.
  • Antibodies can be used to inhibit the polymerase prior to amplification.
  • the antibodies are specific for the thermostable DNA polymerase, inhibit the enzymatic activity of the DNA polymerase at temperatures below about 5O 0 C and are deactivated at higher temperatures.
  • Useful antibodies include monoclonal antibodies, polyclonal antibodies and antibody fragments.
  • the antibody is monoclonal.
  • Antibodies can be prepared using known methods such as those described in Harlow et al., Antibodies: A
  • PCR reagents have been supplied, thermal cycling can be achieved using a heating device and controller. PCR reactions can be multiplexed to assay for several target nucleic acids simultaneously.
  • standard PCR methods may be use.
  • qPCR, reverse-transcription PCR, or multiplex PCR methods may be used.
  • qPCR quantitative PCR
  • real-time PCR a method of polymerase chain reaction (PCR) where a target nucleic acids can be simultaneously amplified and quantified.
  • PCR polymerase chain reaction
  • a positive reaction is detected by accumulation of a fluorescent signal.
  • the C T cycle threshold
  • Cj levels are inversely proportional to the amount of target nucleic acid in the sample (Le. the lower the C T level the greater the amount of target nucleic acid in the sample).
  • Another aspect of the disclosure is directed to isothermal amplification methods to detect target nucleic acid, wherein the method relies on the target nucleic acid-dependent amplification of signal from a detectable label bound to a nucleic acid probe.
  • Methods of isothermal amplification are described in PCT Application No. PCT/NZ2007/000197.
  • Isothermal amplification can be by strand displacement amplification, rolling circle
  • NCR Nuclease Chain Reaction
  • RNCR RNAse-mediated Nucleases Chain Reaction
  • Both of these methods replace strand displacement with the selective degradation of one of the strands of DNA.
  • the process can be initiated by using restriction endonucleases or RNAse H when one of the strands contains ribonucelotides.
  • the Polymerase Nuclease Chain Reaction (PNCR) relies on nuclease cleavage in the presence of target DNA followed by an extension process using a DNA polymerase, RNAse-Mediated Detection (RMD) which is a method of strand degradation by RNAse H on DNArRNA hybrids.
  • RMD is an effective linear amplification system that is sometimes used in combination with other methods.
  • Tandem Repeat Restriction Enzyme Facilitated (TR-REF) Chain Reaction or Inverted reverse Complement Restriction Enzyme Facilitated (IRC-REF) Chain Reaction are two variants of a method that relies on the cyclical production of a detector probe that contains tandem repeats. These repeats are copied by a DNA polymerase when a specific oligonucleotide trigger can act as a primer. Next, restriction endonucleases attack the newly formed double-stranded DNA and this releases the original primer and a second primer so that two new cycles can be initiated. Isothermal amplification reactions can be multiplexed to assay for several target nucleic acid sequences of interest simultaneously.
  • PNAs Peptide Nucleic Acids
  • PNAs Peptide Nucleic Acids
  • PNAs Peptide Nucleic Acids
  • the use of target-binding regions comprising PNAs is particularly contemplated in circular probes, where, prior to the formation of the target probe hybrid, the target-binding region of the probe may be substantially double-stranded.
  • target-binding domain and its equivalent “target binding region” refer to nucleic acid sequence present in a nucleic acid molecule that is sufficiently
  • target probe hybrid complementary to nucleic acid sequence present in the target nucleic acid to allow the hybridisation of the target-binding region and the target nucleic acid, and so to form a target probe hybrid.
  • SNP Single Nucleotide Polymorphism
  • a demonstration of the importance of the field is the International HapMap Project, whose goal is to develop a complete haplotype map of the human genome.
  • Single Nucleotide Polymorphisms can provide genetic markers for specific traits and diseases.
  • a number of successful platforms and technologies are available for identifying the allele at a defined SNP locus.
  • Single SNP's can be interrogated by a variety of polymerase-based techniques such as PCR and primer extension. In some cases, the products of a primer extension system are analyzed in multiplex using mass spectroscopy.
  • oligonucleotides to polymorphic regions of the genome. Examples of the latter approach are the Affymetrix high density arrays.
  • the methods for amplifying target nucleic acids may be reliant on detecting or measuring the signal from a label, preferably the light emission of a probe labelled with a light-emitting label.
  • label refers to any atom, molecule, compound or moiety which.can be attached to a nucleic acid, and which can be used- either to provide a detectable signal or to interact with a second label to modify the detectable signal provided by the second label.
  • Preferred labels are light-emitting compounds which generate a detectable signal by fluorescence, chemiluminescence, or bioluminescence.
  • Still more preferred labels are light-emitting compounds the signal of which is diminished or rendered undetectable when in sufficiently close proximity to a masking group, for example, a quenching chromophore.
  • nucleic acid amplification may be detected by fluorescence.
  • Light emitting labels may be used in PCR and isothermal detection methods.
  • Mechanisms by which the light emission of a compound can be quenched by a second compound are described in Morrison, 1992, in Nonisotopic DNA Probe Techniques (Kricka ed., Academic Press, Inc. San Diego, Calif.), Chapter 13. Mechanisms may include fluorescence energy transfer (FRET), non-radiative energy transfer, long-range energy transfer, dipole-coupled energy transfer, and Forster energy transfer.
  • FRET fluorescence energy transfer
  • the primary requirement for FRET is that the emission spectrum of one of the compounds, the energy donor, must overlap with the absorption spectrum of the other compound, the energy acceptor. Styer and Haugland, 1967, Proc. Natl. Acad. Sci. U.S.A.
  • Exposure of a detection sequence means the detection sequence is rendered accessible for detection, for example accessible for binding to a detection probe. Conversely, the terms "hidden” or “masked” and their grammatical equivalents mean that the element(s) in respect of which these terms are used is/are not accessible.
  • a detection sequence may be hidden or masked when bound to nucleic acid molecule other than a detection probe.
  • hybridization and grammatical equivalents refers the formation of a multimeric structure, usually a duplex structure, by the binding of two or more single-stranded nucleic acids due to complementary base pairing.
  • Alternative labelling systems can be also be used that demonstrate the cleavage of a label from moiety that can be bound to a solid matrix.
  • An example would be a biotin label that could be bound to immobilised avidin and thus non-cleavage of the probe would bind a secondary label present on the other end of the probe.
  • Such a method would have applications for dipstick-based detection.
  • Yet more detection system may use labels that can be distinguished by nanopore technology. The methods described herein are applicable to the detection of probes labelled with a single label, although multiple labels may be employed.
  • Detection of the cleaved probe occurs when the label, for example a fluorophore, is sufficiently removed from the masking group, for example a quencher, by the cleavage event, or the probe-denaturing process the cleavage event allows. This diminishes the interaction of the masking group and the label • and so allows emission of the signal.
  • the term "masking group” means any atom, molecule, compound or moiety that can interact with the label to decrease the signal emission of the label. The separation of label and masking group resulting from the cleavage event or the probe-denaturing process the cleavage event allows in turn results in a detectable increase in the signal emission of the attached label.
  • signal emission ⁇ may include light emission, particle emission, the appearance or disappearance of a colored compound, and the like.
  • chromophore refers to a nonradioactive compound that absorbs energy in the form of light. Some chromophores can be excited to emit light either by a chemical reaction, producing chemiluminescence, or by the absorption of light, producing fluorescence.
  • fluorophore refers to a compound which is capable of fluorescing, i.e. absorbing light at one frequency and emitting light at another, generally lower, frequency.
  • bio luminescence refers to a form of cherniluminescence in which the light-emitting compound is one that is found in living organisms.
  • bioluminescent compounds include bacterial luciferase and firefly luciferase.
  • quenching refers to a decrease in fluorescence of a first compound caused by a second compound, regardless of the mechanism. Quenching typically requires that the compounds be in close proximity. As used herein, either the compound or the fluorescence of the compound is said to be quenched, and it is understood that both usages refer to the same phenomenon.
  • fluorophores and chromophores described in the art are suitable for use in the methods presently disclosed. Suitable fluorophore and quenching chromophore pairs are chosen such that the emission spectrum of the fluorophore overlaps with the absorption spectrum of the chromophore. Preferably, the fluorophore would have a high Stokes shift (a large difference between the wavelength for maximum absorption and the wavelength for maximum emission) to minimize interference by scattered excitation light.
  • Suitable labels which are well known in the art include, but are not limited to, fluorescein and derivatives such as FAM, HEX, TET, and JOE; rhodamine and derivatives such as Texas Red, ROX, and TAMRA; Lucifer Yellow, and coumarin derivatives such as 7-Me2N- coumarin-4-acetate, T-OH- ⁇ -CH.S-coumarin-S-acetate, and 7-NH2-4-CH3-coumarin-3-acetate (AMCA).
  • fluorescein and derivatives such as FAM, HEX, TET, and JOE
  • rhodamine and derivatives such as Texas Red, ROX, and TAMRA
  • Lucifer Yellow and coumarin derivatives
  • 7-Me2N- coumarin-4-acetate T-OH- ⁇ -CH.S-coumarin-S-acetate
  • 7-NH2-4-CH3-coumarin-3-acetate AMCA
  • FAM, HEX, TET, JOE, ROX, and TAMRA are marketed by Perkin Elmer, Applied Biosystems Division (Foster City, Calif.)- Texas Red and many other suitable compounds are marketed by Molecular Probes (Eugene, Oreg.). Examples of chemiluminescent and
  • bioluminescent compounds that may be suitable for use as the energy donor include luminol
  • the detectable label be a light- emitting label and the masking group be a quencher, such as a quenching chromophore
  • the label may be an enzyme and the masking group an inhibitor of said enzyme. When the enzyme and inhibitor are in sufficiently close proximity to interact, the inhibitor is able to inhibit the activity of the enzyme. On cleavage or denaturation of the probe, the enzyme and inhibitor are separated and no longer able to interact, such that the enzyme is rendered active.
  • the methods for deactivating contaminating nucleic acids, extracting target nucleic acid from a sample, and amplifying target nucleic acid described herein are compatible and may be combined in a single closed system.
  • the closed system comprises a single vessel or tube.
  • the reagents for deactivating contaminating nucleic acids, extracting target nucleic acid from a sample, and amplifying target nucleic acid are provided in a manner allowing the system to remain closed after deactivation of contaminating nucleic acids.
  • compatible reagents are provided in a single mixture and then the system is closed.
  • the reagents can be functionally sequestered and provided sequentially.
  • the nucleic acid deactivating reagent is compatible with the nucleic acid extracting reagent (e.g. thermophilic proteinase) and can be added in a single mixture, hi a non-limiting example a mesophilic nucleic acid modifying deactivating reagent is active at about 25-40°C and inactive at about 65-8O 0 C, while the thermophilic proteinase is inactive at about 25-40°C and active at about 65-80 0 C.
  • the nucleic acid extracting reagent e.g. thermophilic proteinase
  • the deactivating reagent and nucleic acid extracting reagent are also compatible with nucleic acid amplification reagents and can be provided in a single mixture
  • the nucleic acid amplification reagents may be unaffected by the enzymes and the process for deactivating contaminating nucleic acids and extracting the target nucleic acid.
  • the DNA polymerase may be resistant to proteolytic cleavage by
  • thermophilic proteinase thermophilic proteinase
  • the nucleic acid deactivating reagent may be provided in combination with the extraction and amplification reagents.
  • the deactivating reagent may be provided in combination with a PCR master mix, containing all the required components to carry out a PCR reaction (buffer containing deoxyribonucleotides, divalent ions, oligonucleotide primers, and DNA polymerase) except the target DNA to be amplified.
  • the reagents may be functionally sequestered in a closed system and provided sequentially.
  • some DNA polymerases such as Tag DNA polymerase are degraded by the thermophilic proteinase in the extraction reagents, so post- extraction delivery strategies for the polymerase should be considered.
  • Possible strategies may include: (1) delivery of the polymerase and any other sensitive reagents after the deactivation and extraction processes are complete. This may be a delivery via an inlet port by microfluidics or a solid dispenser.
  • the polymerase and other sensitive reagents can be added to the deactivation and extraction reagents in a protected form. This can be in the form of a bead or film with the sensitive reagents microencapsulate within.
  • the polymerase can be modified to protect it from the thermophilic proteinase for example by the attachment of antibodies.
  • Novel polymerases can be used that are resistant to proteolytic cleavage.
  • the amplifying reagents are added by microfluidics or a solid dispenser.
  • the amplifying reagents are added by microcapsules comprising the amplifying reagents.
  • the microcapsules are pre-disposed in the vessel or tube in which the amplification will be carried out.
  • the microcapsules are heat-labile capsules.
  • the heat-labile capsules are agarose or wax beads.
  • the heat-labile capsules release amplification detecting reagents when exposed at a temperature sufficient to melt or dissolve the capsules.
  • EMA fluorescence should be taken into account for the quencher and/or reporter dyes used with quantitative PCR.
  • the closed system comprising a single vessel or tube for deactivating contaminating nucleic acids, extracting target nucleic acid, and amplifying target nucleic acid may be a device.
  • Figure 2 illustrates a non-limiting example of a preferred embodiment of a device.
  • the preferred device may include an external stimulus source for converting the nucleic acid deactivating reagent from an inactive form to an active form.
  • a source of specific narrow spectrum wavelength light, broad spectrum white light, or UV light activates EMA by causing photolysis to occur.
  • the preferred device would also allow for closed-system reactions, thus requiring little more than simple physical modulation of a reaction between sample insertion and result generation.
  • light and temperature are used to initiate and stop sequential chemical reactions allowing multi- step procedures to be performed without complex pumps, valves or microfluidics.
  • Light and heat can be controlled by many simple devices including microelectronics, LEDs, Peltier plates or incandescent light bulbs.
  • the device may be compatible with reaction conditions for all stages of the combined method, deactivating contaminating nucleic acids, to extracting target nucleic acids from a sample, to amplifying the target nucleic acid.
  • This system can be integrated with existing technologies.
  • the device includes a single chamber.
  • the chamber is dark or light-tight.
  • the chamber holds an externally supplied tube (e.g. a PCR tube) or plate (e.g. a 96- well microtiter plate), which is placed within the device,
  • the device may comprise an inlet port, an outlet port, a chamber, a detector for emitted fluorescence, and an excitation light source.
  • the device may further comprise a means for controlling the temperature within the chamber.
  • the device also comprises a light source for activating a nucleic acid deactivating reagent.
  • the deactivating reagent- activating light source may be an incandescent lamp, a fluorescent lamp, or an array of light emitting diodes.
  • the device further includes microfluidics, microchips, nanopore technologies and miniature devices.
  • the device or components of the device may be disposable.
  • the device may also be hand-held.
  • the present described devices and methods may have applications for a range of nucleic acid diagnostic techniques where clean-up of nucleic acids to remove contaminants is particularly beneficial, or for diagnostic techniques where the present devices and methods may be adapted to achieve a similar beneficial outcome.
  • Device control can be achieved by standard electronic methods using hardware, software and firmware typical of thermal cycling devices. Likewise, any integrated detection system could use similar programmable devices.
  • Data produced by the detection device may range from a simple yes/no detection when the device is used for detecting a specific agent to real-time data where the time is measured for the signal to reach a pre-defined threshold thereby giving quantitative data.
  • electrophoretic data could be produced in the form the taken for peaks of fluorescence to reach a detector placed at a point along a capillary electrophoresis device.
  • Data analysis can be achieved using a computer program supplied to the device either via and external electronic port, wireless technology, an internal storage device or internal firmware.
  • reporting may be in the form of any visible indicator such as a light or and LCD or LED display.
  • the raw data, processed data or partially processed data can be transferred to an external computer via any form of removable storage device or a communications cable.
  • the results can utilize wireless technology to obtain data base information or use database information stored on the device that may aid in the identification of target nucleic acid present in the sample.
  • Results can be binary, i.e. present or not present, or they can be quantitative or multivariate.
  • Example 1 Use of ethidium monoazide in a combined nucleic acid amplification process
  • the experiment was performed on a dilution series of Escherichia coli cells, and the presence of the cells was detected with universal 16S rRNA oligonucleotide primers. These primers are typical of the type used in microbial analysis. Their advantage is that they can be used for any known bacterial species; their disadvantage is that they also detect any contaminating bacterial DNA.
  • the purposes of the experiment were to demonstrate that reagent purification, DNA extraction and DNA amplification by qPCR could be carried out in a single sealed vessel using only light and heat to control the reaction; and to demonstrate a significant improvement in the lower detection limits achieved by this process.
  • the qPCR primers had the following sequences:
  • the Quanta Bioscience qPCR reagents include dNTP's, buffer, MgCl 2 , and Taq DNA polymerase.
  • Escherichia coli MGl 655 cells were grown overnight in LB broth. The cells were then centrifuged at 12,000RCF for 5 minutes and resuspended in water to a cell density of 2x10 7 cells per ml. This density is the equivalent of 10 D cells per 5 ⁇ l. Cells were then diluted in a 1 : 10 serial dilution in ultra-pure water to approximately 10 cells per 5 ⁇ l. Following the serial dilution, 5 ⁇ l of each dilution was placed into 8 wells of an optically transparent 96- well microtiter plate.
  • FIG. 3 shows C T values obtained for qPCR reactions performed in the absence of EMA. The results demonstrate that extraction and detection can be performed in a single reaction vessel without opening the tube. As can be seen by the dashed line, the highest Cj level that can be obtained is approximately 25 cycles. This is the Cj value of the negative control (ultra-pure water only). This Cj value equates to approximately 1,000 cells (genomes).
  • Figure 4 shows improved results when EMA treatment is used. EMA treatment is integrated into the entire reaction. Here, the negative control C T has been raised to
  • This experiment demonstrates that all steps in a combined process including deactivation of contaminating nucleic acids, extraction of target DNA, and PCR amplification of target DNA can be performed in a single, sealed vessel. AU reagents were simultaneously treated, as was the reaction vessel in which they were contained. This experiment also demonstrates the unexpectedly significant effect of including EMA treatment to deactivate contaminating nucleic acids in a closed system. As shown in Figure 4, adding EMA to a negative control, amplification cycles can be increased to approximately 37 cycles before contaminating DNA is detected. It was unexpected that combination of all three stages in the process of decontamination, extraction and detection into a single, closed-tube procedure, would be possible given that the reagents required for each step are intuitively incompatible.
  • One advantage of this new method is that the integration of the contaminating nucleic acid deactivation step with the extraction and amplification steps has improved detection level by more than three orders of magnitude. This enhancement has been achieved by reducing the level of background noise, thereby allowing more amplification cycles to be used.
  • Maintaining a closed tube system for all stages of the reaction also represents a significant enhancement in securing the sample from contamination.
  • integration of the deactivation step into the reaction allows all reagents and the reaction vessel to be simultaneously processed. This simplifies the procedure and enables easy automation.
  • the integration of the deactivation step with the extraction step also allows for the detection of DNA from living cells and excludes DNA from contaminated reagents, sample matrix, dead cells or plastic-ware.
  • Example 2 Comparison of combined EMA treatment, nucleic acid extraction and amplification and EMA pre-treatment
  • the combined method is performed as described in Example 1 above.
  • the EMA pre-treatment method is performed as follows: 0.1 ⁇ g/ ⁇ l of EMA is added to samples in a 96-well microtiter plate and sealed with a transparent adhesive lid and is held at 4°C for 5 minutes in the dark. The plate is then exposed through the seal to a 600W halogen lamp at a distance of 200 mm from the plate for 5 minutes with the microtiter plate maintained at 4 0 C.
  • the transparent adhesive lid is removed from the 96-well microtiter plate and Quanta PCR mix, Primer 1, Primer 2, and EAl proteinase lU/ ⁇ l are added to each sample.
  • the 96-well microtiter plate is then re-sealed with a transparent adhesive lid and placed in an Applied Biosystems 7300 Real-time PCR System. The qPCR is performed as described in Example 1 above.
  • Example 3 Use of a mesophilic nucleic acid modifying enzyme in a combined nucleic acid amplification process
  • a combined nucleic acid amplification process incorporating the use of a mesophilic nuclease for deactivating contaminating nucleic acids, and a thermophilic proteinase that is inactive on the DNA polymerase and can be used in combination with the deactivating nucleases by virtue of different temperature activation profiles.
  • thermophilic proteinase for extracting target nucleic acid, providing that the thermophilic proteinase has low activity on the amplification enzyme(s) used in the qPCR;
  • necessary reagents for the qPCR e.g. dNTP's, buffer, MgCI2, oligonucleotide primers, and Taq DNA polymerase.
  • thermophilic proteinase has low activity at this temperature and does not affect the mesophilic nuclease.
  • thermophilic proteinase the proteinase simultaneously digests the mesophilic nuclease and mediates the lysis and degradation of the cellular material to release the target nucleic acid.
  • thermophilic proteinase 4. incubate the reagent cocktail at about 90 to 95°C to simultaneously activate the Taq DNA polymerase and inactivate the thermophilic proteinase.
  • Example 4 A device with a controlled light source
  • Figure 2 illustrates one embodiment of the combined contaminating nucleic acid deactivation, nucleic acid extraction, and nucleic acid amplification process performed using a device.
  • the device is a sealed unit with a light source for exposing a 96-well microtiter plate to light at a wavelength of 510nm that can, for example, activate the nucleic acid deactivating reagent ethidium monoazide (EMA) and lead to the covalent linkage of EMA with
  • One purpose of the device is to simplify the application of EMA in a secure chamber that protects the sample from (i) extraneous ambient light, (ii) further contamination with nucleic acids, and (iii) protects the user from an intense light source.
  • the device of Figure 2 has dimensions suited for the 96-well microtiter plate, and consists of a light-tight (or partially light-tight) chamber with an array of light emitting diodes (LED's) that exposes the 96-well microtiter plate to light at a wavelength of 510nm, a timer that controls the dark incubation period and the light period, and a temperature-controlled block (Peltier Device, also known as a Termoelectric Cooler or TEC) for maintaining the 96-well microtiter plate at a desired temperature.
  • LED's light emitting diodes
  • TEC Termoelectric Cooler
  • wavelength specific LED's are that less thermal energy is released resulting is less heating of the samples, less unnecessary wavelengths are used which also results in less heating, a lower power source can be used for the device, and will result in less photo-bleaching of fluorescent reagents. Furthermore, using an array of LED's ensures that all wells in the 96-well microtiter plate receive an equivalent amount of light.
  • the device of Figure 2 is used, for example, to deactivate contaminating nucleic acids in qPCR reagents.
  • the combined nucleic acid amplification processes utilizing ethidium monoazide or mesophilic nucleases described in the above examples is used with the device of Figure 2.
  • the device in conjunction with a nucleic acid deactivating agent has a particular advantage in qPCR.
  • Such a device could become an integrated part of a thermal cycler or a real-time PCR machine thereby creating a single piece of hardware capable of carrying out all of the steps from decontamination, through nucleic acid extraction, to amplification and detection.
  • photoreactive analogues spectroscopic analysis of deoxyribonucleic acid binding properties. Biochemistry 20(7), 1887-1892.
  • Taq polymerase contains bacterial DNA of unknown origin. Molecular and Cellular Probes 4, 445-450.

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Abstract

L'invention concerne un procédé utilisant des réactifs de désactivation de l'acide nucléique pour la désactivation d'acides nucléiques contaminants, et des protéinases thermophiles pour l'extraction d'acides nucléiques dans un système fermé, ce procédé étant destiné à être utilisé en tandem avec des procédés d'amplification d'acides nucléiques cibles présents dans un échantillon. Le procédé combiné permet d'utiliser des dispositifs de régulation de température simplifiés de façon à effectuer un test simple et précis, hors laboratoire, dans une grande variété d'applications dans les domaines de la médecine, l'industrie, l'environnement, le contrôle qualité, la sécurité et la recherche.
PCT/NZ2010/000137 2009-07-02 2010-07-02 Blocage, extraction et détection combinés d'acide nucléique dans un réceptacle de réaction unique WO2011002319A2 (fr)

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JP2012518502A JP2012531907A (ja) 2009-07-02 2010-07-02 単一の反応槽において組合せられた、核酸ブロッキング、抽出、及び検出
EP10794424.1A EP2449125A4 (fr) 2009-07-02 2010-07-02 Blocage, extraction et détection combinés d'acide nucléique dans un réceptacle de réaction unique

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WO2014025500A3 (fr) * 2012-07-16 2015-09-24 Microlab Horizon Llc Extractions à base d'enzyme thermostable sur une puce microfluidique intégrée pour l'analyse biologique
JP2016015738A (ja) * 2009-09-16 2016-01-28 フィッシャー, ジョン, ジェイ.FISCHER, John, J. 標準の携帯通信機器の注意散漫防止及び安全プロトコル
WO2016065300A1 (fr) * 2014-10-24 2016-04-28 Eshoo Mark W Cartouche microfluidique
US9584652B2 (en) 2009-09-16 2017-02-28 Try Safety First, Inc. Standard mobile communication device distraction prevention and safety protocols
CN107988046A (zh) * 2018-01-23 2018-05-04 吉林大学 基于lamp的自吸式多通道病原菌检测微流控芯片
US10370175B2 (en) 2012-07-30 2019-08-06 P.C.O.A. Devices Ltd. Receptacle for containing and dispensing solid medicinal pills
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US11264125B2 (en) 2015-10-15 2022-03-01 Dosentrx, Ltd. Image recognition-based dosage form dispensers
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EP2324128A1 (fr) * 2008-08-14 2011-05-25 Zygem Corporation Limited Procédé de détection d'acide nucléique à température contrôlée adapté pour être mis en pratique dans un système fermé
EP2324128A4 (fr) * 2008-08-14 2012-09-12 Zygem Corp Ltd Procédé de détection d'acide nucléique à température contrôlée adapté pour être mis en pratique dans un système fermé
US11196856B2 (en) 2009-09-16 2021-12-07 Cell Command, Inc. Standard mobile communication device distraction prevention and safety protocols
US10425528B2 (en) 2009-09-16 2019-09-24 Cell Command, Inc. Standard mobile communication device distraction prevention and safety protocols
US11756419B2 (en) 2009-09-16 2023-09-12 Cell Command Inc. Standard mobile communication device distraction prevention and safety protocols
US9584652B2 (en) 2009-09-16 2017-02-28 Try Safety First, Inc. Standard mobile communication device distraction prevention and safety protocols
US9756175B2 (en) 2009-09-16 2017-09-05 Cell Command, Inc. Standard mobile communication device distraction prevention and safety protocols
JP2016015738A (ja) * 2009-09-16 2016-01-28 フィッシャー, ジョン, ジェイ.FISCHER, John, J. 標準の携帯通信機器の注意散漫防止及び安全プロトコル
US10194018B2 (en) 2009-09-16 2019-01-29 Cell Command, Inc. Standard mobile communication device distraction prevention and safety protocols
US10715655B2 (en) 2009-09-16 2020-07-14 Cell Command, Inc. Standard mobile communication device distraction prevention and safety protocols
US10399725B2 (en) 2012-07-05 2019-09-03 P.C.O.A. Devices Ltd. Medication dispenser
WO2014025500A3 (fr) * 2012-07-16 2015-09-24 Microlab Horizon Llc Extractions à base d'enzyme thermostable sur une puce microfluidique intégrée pour l'analyse biologique
US10370175B2 (en) 2012-07-30 2019-08-06 P.C.O.A. Devices Ltd. Receptacle for containing and dispensing solid medicinal pills
US10456332B2 (en) 2014-06-22 2019-10-29 P.C.O.A. Devices Ltd. Controlled dosage form-dispensing system
WO2016065300A1 (fr) * 2014-10-24 2016-04-28 Eshoo Mark W Cartouche microfluidique
US10952928B2 (en) 2015-04-20 2021-03-23 Dosentrix Ltd. Medication dispenser depilling mechanism
US11264125B2 (en) 2015-10-15 2022-03-01 Dosentrx, Ltd. Image recognition-based dosage form dispensers
US11458072B2 (en) 2015-11-02 2022-10-04 Dosentrx Ltd. Lockable advanceable oral dosage form dispenser containers
US12115131B2 (en) 2015-11-02 2024-10-15 Dosentrx Ltd. Lockable advanceable oral dosage form dispenser containers
CN107988046A (zh) * 2018-01-23 2018-05-04 吉林大学 基于lamp的自吸式多通道病原菌检测微流控芯片

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