JP2012531907A - Nucleic acid blocking, extraction and detection combined in a single reactor - Google Patents

Abstract

  The present invention relates to a method using a nucleic acid inactivating reagent for inactivating contaminating nucleic acid and a thermophilic proteinase for extracting target nucleic acid in a closed system used together with a method for amplifying target nucleic acid in a sample. According to the above method, simple and temperature-controlled devices are used for accurate and streamlined inspections in view of their widespread use in medical, industrial, environmental, quality control, security, and research fields It is possible.

Description

Detailed Description of the Invention

〔Technical field〕
The present application generally relates to a method for rapidly amplifying a target nucleic acid contained in a sample, performed in a closed system. More specifically, the present application relates to a method of performing inactivation of contaminating nucleic acid and extraction of target nucleic acid, and can be performed in a closed system device for detecting target nucleic acid in a sample. The present invention relates to a method adaptable to the temperature control amplification method of

[Background Technology]
For treatments in various fields such as medical treatment, industry, environment, security, research, quality control, etc., a treatment that can amplify nucleic acid in a sample accurately and reliably is desirable. Preferably, such treatment is performed in a short time and produces accurate results, and is a closed system, i.e., analyzed to prevent yield loss or accidental contamination with unwanted nucleic acids or nucleases. This is a process that can be executed in a system that does not need to be released during the process. The nucleic acid amplification process can be divided into three stages: inactivation or removal of contaminating nucleic acids, extraction of target nucleic acids, and amplification of target nucleic acids. The main problem in these stages is that these three stages usually require that each stage be executed by a different process.

[Inactivation of contaminating nucleic acids]
Nucleic acid amplification is very sensitive to contamination. The polymerase chain reaction (PCR) can amplify even a small amount of DNA, and its amplification ability is one of the great advantages of the polymerase chain reaction. On the other hand, even such traces of contaminating DNA are co-amplified by such excellent amplification ability. Amplification of contaminating DNA leads to misleading results or results that are ambiguous or misleading (Wilson et al., 1990). In particular, for phylogenetic, evolutionary, and diagnostic studies, target highly conserved, evolutionary and identically sourced eubacterial ribosomal 16S rRNA genes using universally utilized primers This concept frequently results in false positive PCR (Hilali et al., 1997; Corless et al., 2000; Mohammadi et al., 2003). In fact, contamination of commercially available DNA polymerases such as Taq DNA polymerase has been repeatedly reported as one of the most likely causes of DNA contamination by foreign bacteria (Bottger, 1990; Rand et al., 1990; Schmidt et al., 1991; Hilali et al., 1997).

  Furthermore, eubacterial DNA is widely distributed in the environment and can contaminate the PCR mix at virtually any stage of processing via a laboratory technician (Kitchin et al., 1990; Millar et al., 2002). Other causes of DNA contamination include aerosols (Saksena et al., 1991), disposable PCR reagents, plastic containers (Millar et al., 2002), and commercially available PCR primers (Goto et al, 2005). .

  Another area where DNA contamination is a problem is the classification of mitochondrial DNA (mtDNA). Sequence analysis of human mtDNA is widely used for forensic purposes to clarify its characteristics even from specimens for which sufficient nuclear DNA cannot be obtained. Frequently, mtDNA is the only intact DNA left in trace or degraded samples. However, laboratories where these types of samples are processed often have more DNA than the samples contain, and the handling of the samples requires strict quality assurance (Carracedo). et al., 2000).

  Similarly, anthropological or phylogenetic analysis of ancient biological materials has been hampered by the fact that the targeted DNA copy number is low compared to the large amount of mtDNA present in the environment and laboratory. Many reports of such problems can be found in scientific journals (eg, Lourdes Sampietro et al., 2006).

[Decontamination by solution]
The decontamination solution for DNA amplification currently known in the field is by pretreatment using a nucleolytic enzyme or chemical that deactivates nucleic acid contaminants. Examples of enzymes used for reagent processing are DNAseI or Sau3A. Solutions for RNA analysis are often treated using diethyl pyrocarbonate (DEPC), and double-stranded DNA can be removed using ethidium monoazide (New Zealand patent 545,894). However, these DNA decontamination solutions have not been used in combination with closed-system reagent cocktails for target nucleic acid decontamination, extraction, and amplification.

[Nucleic acid extraction]
In isolation of target nucleic acids for diagnostic treatment, it is often necessary to extract nucleic acids from natural drugs. The use of such isolation ranges from forensic DNA fingerprinting to medical, agricultural, anthropological, taxonomic and environmental monitoring. It is important that the nucleic acid sample is not contaminated, especially in situations where the concentration of nucleic acids in the initial sample is very low, or where the results obtained may contain errors due to nucleic acid contamination. It is. This is especially important for trace bacterial samples, ancient samples, and forensic samples where the amount of starting material is in picograms or less. In general nucleic acid extraction techniques, it is necessary to open and close the sample tube at each stage through the extraction procedure, so that the above general nucleic acid extraction techniques have problems when considering the viewpoint of preventing nucleic acid contamination. Yes. Nucleic acid contamination may occur simply as a result of the sample tube being opened to the atmosphere or touched by a technician.

〔amplification〕
In order to confirm the presence or absence of the target nucleic acid in the sample, a method for amplifying the target nucleic acid can be performed. There are many situations where it is desirable to detect small amounts of a specific nucleic acid sequence from a complex mixture, and current nucleic acid detection methods selected for this purpose need to have a high degree of specificity and sensitivity. A simple method capable of directly detecting one nucleic acid molecule in a specific sequence has not been established so far, and all methods currently used include at least one step of amplifying the signal. Yes. The most widely used method for amplifying the signal is PCR. The PCR method uses temperature cycling and a thermostable DNA polymerase to amplify the target nucleic acid logarithmically.

  For all existing treatment methods, the three steps of inactivation, extraction, and amplification of the target nucleic acid are discontinuous with each other, so to move from one step to the next, at some point in the process. The disadvantage is that the tube must be opened, which exposes the sample to contamination with environmental DNA. These disadvantages are particularly important because plastic instruments used in experiments such as pipette tips and tubes are also prone to contamination and contamination of experimental samples is unavoidable, no matter how careful testing means and strict laboratory standards are. Therefore, it is a process that combines inactivation of contaminating nucleic acids, extraction of target nucleic acids, and amplification of target nucleic acids into one closed system, which can identify target nucleic acids accurately and in a short time There is clearly a need for a process that takes place in a tank or apparatus that can. The target nucleic acids correspond in particular to microorganisms encountered in a wide range of situations or mixed groups.

  It is noted that the documents cited in this specification do not constitute prior art. The discussion of the cited documents merely describes the claims of the authors of the documents, and the Applicant has the right to refute the accuracy and appropriateness of the cited documents. Moreover, although many documents are cited in this specification, it should be clearly stated that such cited documents do not form common sense in the technical field to which the present invention belongs.

[Summary of the Invention]
The above methods, processes, and apparatus described herein overcome the above-mentioned drawbacks and disadvantages. In the present specification, a method for combining inactivation of contaminating nucleic acids, extraction of target nucleic acids, and amplification of target nucleic acids into one process that can be performed in a closed system will be described. The method for inactivating contaminating nucleic acids disclosed herein can be controlled by applying an external stimulus and is compatible with most treatments of nucleic acid extraction and nucleic acid amplification. Furthermore, the conditions for nucleic acid extraction by thermophilic proteinase treatment are compatible with most nucleic acid amplification treatment conditions.

One embodiment of the present invention is a method for amplifying a target nucleic acid in a closed system comprising:
i) an addition step of adding a nucleic acid inactivating reagent, a thermophilic proteinase, and a nucleic acid amplification reagent to a sample containing a target nucleic acid to constitute the closed system,
The nucleic acid inactivating reagent has an active form and an inactive form, and only the active form inactivates the contaminating nucleic acid in the closed system, thereby preventing the contaminating nucleic acid from reacting with the nucleic acid amplification reagent. ,
The nucleic acid inactivating reagent changes to the active form only when an external stimulus is applied,
The thermophilic proteinase is in an active form at about 65 ° C. to about 80 ° C. and an inactive form at about 90 ° C. or higher;
ii) a closing step for closing the closed system;
iii) an application step of applying the external stimulus for a time sufficient for the nucleic acid inactivating reagent to change from the inactive form to the active form,
The application prevents the contaminating nucleic acid in the closed system from reacting with the nucleic acid amplification reagent, and the nucleic acid inactivating reagent is applied after the contaminating nucleic acid stops reacting with the nucleic acid amplification reagent and then changes to an inactive form. Process,
iv) an incubation step of incubating the sample at about 65 ° C. to about 80 ° C., wherein the incubation activates a thermophilic proteinase and generates one or more of cell lysis, protein degradation, cell wall enzyme degradation, An incubation step in which target nucleic acids for amplification are extracted;
v) an incubation step of incubating the sample at about 90 ° C. or more, wherein the incubation causes a thermocatalytic proteinase autocatalytic reaction to occur;
vi) an amplification step of amplifying the target nucleic acid,
The closed system provides a method comprising a single tank or tube.

  In other embodiments, the nucleic acid inactivating reagent is brought into the inactive form before incubating the sample at about 65 ° C. to 80 ° C. or simultaneously with incubating the sample at about 65 ° C. to 80 ° C. Change. In yet another embodiment, after the contaminating nucleic acid no longer reacts with the nucleic acid amplification reagent, the external stimulus is applied for a time sufficient for the nucleic acid inactivating reagent to change to the inactive form. The nucleic acid inactivating reagent is changed to the inactive form. In yet another embodiment, after the contaminating nucleic acid no longer reacts with the nucleic acid amplification reagent, the nucleic acid inactivating reagent is changed to the inactive form by removing the external stimulus.

  In still other embodiments, the nucleic acid inactivating reagent is at a concentration sufficient to inactivate contaminating nucleic acids without inhibiting amplification of the thermophilic proteinase, the nucleic acid amplification reagent, or the target nucleic acid. Be prepared.

  In yet another embodiment, the nucleic acid inactivating reagent is a mesophilic nucleic acid modifying enzyme. In still other embodiments, the mesophilic nucleic acid modifying enzyme is a double strand specific endonuclease, a double strand specific exonuclease, a single strand specific nuclease, a restriction endonuclease, RNAses, RNAse H, or an RNA modifying enzyme. It is. In yet another embodiment, the mesophilic nucleic acid modifying enzyme is activated by incubating at about 25 ° C. to 40 ° C. for a time sufficient to change the mesophilic nucleic acid modifying enzyme to the active form. In yet another embodiment, the mesophilic nucleic acid modifying enzyme is inactivated by incubating at about 65 ° C. to 80 ° C. for a time sufficient to change the mesophilic nucleic acid modifying enzyme to the inactive form. .

  In one embodiment of the invention, the nucleic acid inactivating reagent is a nucleic acid intercalating agent. In another embodiment, when the external stimulus is applied, photolysis of the nucleic acid intercalating agent occurs and the nucleic acid intercalating agent changes to the active form. In yet another embodiment, the active form of the nucleic acid intercalating agent is covalently linked to a double stranded nucleic acid. In yet another embodiment, the nucleic acid intercalating agent is ethidium monoazide. In yet other embodiments, the ethidium monoazide is provided at a concentration between about 1 μg / ml and about 5 μg / ml. In yet another embodiment, the ethidium monoazide is provided at a concentration of about 3 μg / ml.

  In one embodiment of the present invention, the external stimulus is specific narrow spectrum wavelength light, broad spectrum white light, or ultraviolet light. In other embodiments, the external stimulus is a specific narrow spectrum wavelength light, broad spectrum white light, or ultraviolet light sufficient to activate the nucleic acid intercalating agent by photolysis of the nucleic acid intercalating agent. It is. In still other embodiments, the external stimulus is sufficient thermal energy to activate the nucleic acid inactivating reagent without substantially activating the thermophilic proteinase. In yet another embodiment, the external stimulus is thermal energy, and the thermal energy causes the temperature of the closed system to be about 25 ° C. for a time sufficient to change the mesophilic nucleic acid modifying enzyme to the active form. Thermal energy sufficient to raise to ~ 40 ° C.

  In one embodiment of the invention, the thermophilic proteinase is EA1.

In another embodiment of the invention, the method comprises
(I) treating the sample with a mesophilic enzyme;
(Ii) further comprising incubating the sample at a temperature lower than about 40 ° C. for a time sufficient to remove the cell wall from the cells.

  In yet another embodiment of the invention, the mesophilic enzyme is cellulase or lysozyme.

  In one embodiment of the invention, the nucleic acid amplification is detected by a fluorophore. In another embodiment, a PCR detection method is performed in the amplification step of the target nucleic acid. In still other embodiments, the PCR detection method is qPCR, multiplex PCR, or reverse transcription PCR.

  In yet another embodiment, an isothermal detection method is performed in the amplification step. In still other embodiments, the isothermal detection method is a strand displacement amplification method, a rolling circle amplification method, a loop-mediated isothermal amplification method, a chimeric primer-derived nucleic acid isothermal amplification method, a Q-beta amplification system, or a OneCutEventAmplificatioN. In still other embodiments, the isothermal detection method comprises nuclease chain reaction (NCR), RNAse mediated nuclease chain reaction (RNCR), polymerase nuclease chain reaction (PNCR), RNAse mediated detection (RMD), tandem repeat restriction enzyme promotion. Techniques such as (TR-REF) chain reaction or reverse reverse complement restriction enzyme promoted (IRC-REF) chain reaction are utilized.

  In one embodiment of the present invention, SNP detection analysis is performed in the amplification step.

  In another embodiment of the present invention, the nucleic acid amplification step is automated.

  In one embodiment of the invention, the nucleic acid amplification reagent is added using a microfluidic or solid dispenser.

  In another embodiment of the present invention, the nucleic acid amplification reagent is added using a microcapsule containing the nucleic acid amplification reagent. In yet another embodiment, the microcapsules are pre-supplied to the tank or tube. In yet another embodiment, the microcapsule is a heat-resistant capsule. In yet another embodiment, the heat-sensitive capsule is an agarose or waxy bead. In still other embodiments, the detection reagent is released from the heat-sensitive capsule when the heat-sensitive capsule is exposed to a temperature sufficient to melt or decompose the heat-sensitive capsule.

  In one embodiment of the present invention, the nucleic acid amplification reagent is resistant to proteolysis by the thermophilic proteinase.

  In other embodiments of the invention, the sample is blood, urine, saliva, semen, excreta, tissue, swabs, tears, bones, teeth, hair, or mucus. In yet other embodiments, the sample is a bacterium, fungus, archaea, eukaryote, protozoan, or virus. In yet other embodiments, the sample is salt water, fresh water, ice, soil, waste, or food.

  In one embodiment of the invention, the tank or tube is a device. In other embodiments, the device is a handheld device. In still other embodiments, the device or a component of the device is disposable. In yet another embodiment, the apparatus comprises an inlet, an outlet, a chamber, a detector that detects the emitted fluorophore, and an excitation light source. In yet another embodiment, the device further comprises a microfluidic, a microchip, a nanopore technology, and a manipulator device.

  In one embodiment of the present invention, the apparatus comprises a sealed unit, a light shielding chamber, a nucleic acid inactivating reagent activation light source, a detector for detecting the emitted fluorophore, and an excitation light source. In another embodiment, the nucleic acid inactivating reagent activation light source is an incandescent lamp, a fluorescent lamp, or a light emitting diode array.

Another embodiment of the present invention is a method for amplifying a target nucleic acid in a closed system comprising:
i) an addition step of adding the mesophilic nucleic acid modifying enzyme, EA1 proteinase, and PCR amplification reagent to the sample containing the target nucleic acid to constitute the closed system,
The mesophilic nucleic acid modifying enzyme has an active form and an inactive form, and only the active form inactivates the contaminating nucleic acid in the closed system, thereby preventing the contaminating nucleic acid from reacting with the PCR amplification reagent,
The mesophilic nucleic acid modifying enzyme is in an active form at about 25 ° C to about 40 ° C, and is in an inactive form at about 65 ° C to about 80 ° C.
The EA1 proteinase is in an active form at about 65 ° C. to about 80 ° C. and an inactive form at about 90 ° C. or higher;
ii) a closing step for closing the closed system;
iii) an incubation step in which the sample is incubated at a temperature of about 20 ° C. to about 40 ° C. for a certain period of time, wherein the mesophilic nucleic acid modifying enzyme is activated by the incubation, and the contaminating nucleic acid in the closed system reacts with the PCR amplification reagent. An incubating step to avoid,
iv) an incubation step in which the sample is incubated at a temperature of about 65 ° C. to about 80 ° C. for a certain period of time, wherein the incubation deactivates the mesophilic nucleic acid modifying enzyme, activates the EA1 proteinase, cell lysis, proteolysis, An incubation step in which target nucleic acids for amplification are extracted by the occurrence of one or more of the degradation of cell wall enzymes;
v) an incubation step of incubating the sample at about 90 ° C. or higher, wherein the incubation causes an autocatalytic reaction of the EA1 proteinase;
vi) an amplification step of amplifying the target nucleic acid,
The closed system provides a method comprising a single tank or tube.

Yet another embodiment of the invention is a method of amplifying a target nucleic acid in a closed system, comprising:
i) an addition step of adding ethidium monoazide, EA1 proteinase, and PCR amplification reagent to a sample containing the target nucleic acid to constitute the closed system,
The ethidium monoazide has an active form and an inactive form, and only the active form inactivates the contaminating nucleic acid in the closed system, thereby preventing the contaminating nucleic acid from reacting with the PCR amplification reagent,
The ethidium monoazide changes to the active form only when photolysis of ethidium monoazide occurs when narrow spectrum wavelength light, broad spectrum white light, or ultraviolet light is applied.
The EA1 proteinase is in an active form at about 65 ° C. to about 80 ° C. and an inactive form at about 90 ° C. or higher;
ii) a closing step for closing the closed system;
iii) an application step of applying the narrow spectrum wavelength light, broad spectrum white light, or ultraviolet light for a first time sufficient to change the ethidium monoazide from the inactive form to the active form. The irradiation prevents contaminating nucleic acids in the closed system from reacting with the nucleic acid amplification reagent, and the ethidium monoazide becomes inactive with respect to the subsequent nucleic acid by photolysis of the ethidium monoazide by the irradiation. Process,
iv) an incubation step in which the sample is incubated at a temperature of about 65 ° C. to about 80 ° C. for a second time, wherein the incubation activates the EA1 proteinase, and one of cell lysis, proteolysis, and cell wall enzyme degradation An incubation step in which the target nucleic acid for amplification is extracted by more than one occurrence;
v) an incubation step of incubating the sample at about 90 ° C. or higher, wherein the incubation causes an autocatalytic reaction of the EA1 proteinase;
vi) an amplification step of amplifying the target nucleic acid,
The closed system provides a method comprising a single tank or tube.

[Brief description of the drawings]
FIG. 1 is a schematic diagram of complex nucleic acid detection processing.

  FIG. 2: A single chamber apparatus provided with a light emitting diode array for inactivating contaminating nucleic acids, extracting target nucleic acids, and amplifying target nucleic acids using an encapsulating reagent.

Figure 3: If the DNA extraction and qPCR was performed in a single vessel is a graph showing the C _T values obtained by qPCR reactions to various Escherichia coli cell counts. In this reaction treatment, no ethidium monoazide treatment was performed. Dashed line in the drawing shows a C _T values obtained when performing qPCR reactions against water only (cell number zero). The error bar is a standard deviation of ± 1 from the average value.

Figure 4: When the DNA extraction and qPCR was performed in a single vessel is a graph showing the C _T values obtained by qPCR reactions to various Escherichia coli cell counts. In this reaction treatment, ethidium monoazide treatment was performed as part of the sequential reaction in the closed tube. Dashed line in the drawing shows a C _T values obtained when performing qPCR reactions against water only (cell number zero). The error bar is a standard deviation of ± 1 from the average value.

[Detailed explanation]
[Definition]
As used herein, the term “nucleic acid inactivating reagent” refers to a chemical, molecule, enzyme, or other biomolecule capable of deactivating a nucleic acid for nucleic acid amplification processing.

  As used herein, the term “inactivation” refers to a process that removes or degrades contaminating nucleic acids, or a process that renders contaminating nucleic acids non-reactive with nucleic acid amplification processes.

  The term “non-reactive” as used herein refers to a nucleic acid state that is not amplified by any nucleic acid amplification step or method. An example of an inactivation reagent that causes such nucleic acid dereactivity includes, but is not limited to, ethidium monoazide that binds to contaminating nucleic acids. In addition, although ethidium monoazide exists in a solution, it is not detected by the signal detection method performed after the target nucleic acid amplification process.

〔Overview〕
The method for amplifying a target nucleic acid is divided into three steps: an inactivation step of contaminating nucleic acid, a target nucleic acid extraction step, and a target nucleic acid amplification step. A system that performs these steps requires different instruments for each process, and adopts a moving method for transferring a sample from one internal instrument to another. The above-described conventional technology discloses that nucleic acid pretreatment, nucleic acid extraction, and nucleic acid amplification are performed in independent processes. In addition, when performing each said process in each closed system, since the said closed system is open | released between each process, it exposes to a potential contamination risk.

  Since the method for inactivating contaminated nucleic acids according to the present invention can be controlled by an external stimulus and is compatible with nucleic acid extraction methods and amplification methods, it can be provided in one closed system together with these methods. In a closed system, a sample containing a target nucleic acid, a nucleic acid inactivating reagent, a nucleic acid extraction reagent, and a nucleic acid amplification reagent are supplied to an open container or open tube, and then the open container or open tube prevents contamination. To be sealed. In an open system, the open container or the open tube is opened for a period sufficient to additionally introduce a reagent such as a nucleic acid extraction reagent or a nucleic acid amplification reagent. For example, the open system is opened at the next timing. That is, the open container or the open tube is opened after the nucleic acid inactivation treatment, before the nucleic acid extraction treatment or after the treatment, or before the amplification treatment.

  Unexpectedly, when the inactivation of contaminating nucleic acid is combined with extraction and amplification of the target nucleic acid in a single closed system, the nucleic acid growth treatment can be performed without using the inactivating reagent for contaminating nucleic acid. The detection sensitivity of nucleic acid amplification was improved several times compared to the case where it was performed.

  According to a preferred embodiment, when a nucleic acid inactivating reagent is used, the detection accuracy of nucleic acid amplification is at least about 2 times, at least about 3 times, at least 4 times compared to when no nucleic acid inactivating reagent is used. At least about 5 times, at least about 8 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 50 times, at least about 1000 times, at least about 250 times, at least about 500 times About at least 750 times, at least about 1000 times, at least about 2000 times, at least about 3000 times, at least about 4000 times, at least about 5000 times, or more.

  According to another preferred embodiment, the nucleic acid amplification detection sensitivity of a method carried out in a closed system using a nucleic acid inactivating reagent is at least twice as high as that of the same method carried out in an open system, At least about 3 times, at least about 4 times, at least about 5 times, at least about 8 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 50 times, at least about 1000 times, It is improved at a rate of at least about 250 times, at least about 500 times, at least about 750 times, at least about 750 times, at least about 1000 times, at least about 2000 times, at least about 3000 times, or more.

  Similar to the nucleic acid amplification method, nucleic acid extraction via the thermophilic proteinase treatment disclosed herein is temperature controlled. In addition, the conditions required for thermophilic treatment are compatible with the conditions required for most amplification processes. Therefore, a method combining inactivation of contaminating nucleic acids, extraction of target nucleic acids, and amplification of the extracted target nucleic acids can be performed in one container or apparatus. The apparatus is also simplified to provide a source of external stimulus that activates the inactivating reagent for contaminating nucleic acids, and a heating / cooling mechanism that treats the original sample material to produce detectable nucleic acid amplification. , Can be a container provided. It is easy to provide a detector in the above apparatus.

  According to the above-described method disclosed in the present specification, it is possible to realize a combined treatment of inactivation of contaminating nucleic acid, extraction of target nucleic acid, and amplification of target nucleic acid, and installation of a pump or use of a microfluid. It is also possible to realize an apparatus that does not require the above. Note that the above-described method may be applied to more complicated subsequent processing as necessary. Moreover, the said process can be performed in the closed system apparatus using an external stimulus and a heating control reaction chain. Nucleic acid inactivating reagents and thermophilic proteinases are used in conjunction with techniques for amplifying nucleic acids using PCR or isothermal amplification, and the nucleic acid inactivating reagents are used to suppress contaminating nucleic acids in the system. The thermophilic proteinase is used to extract nucleic acid contained in the sample. Combining a nucleic acid inactivating reagent that is activated by an external stimulus and then inactivated, with temperature control using a mixture of temperature dependent enzymes, or temperature controlled release of the encapsulated reagent. This simplifies the design of current nucleic acid diagnostic methods and devices. Further, by including an inactivation step as part of the above process, the probability of false positives is reduced, and the nucleic acid amplification sensitivity and detection sensitivity are improved. Simplifying the design of nucleic acid diagnostic methods and devices reduces the failure rate and cost associated with design complexity. The above technique has the further advantage that it can be modified and multiplexed so that multiple types of target nucleic acids contained in a mixed sample can be identified simultaneously.

  FIG. 1 outlines an embodiment of the above-described processing disclosed in detail in this specification. First, a nucleic acid inactivating reagent, a thermophilic proteinase, a nucleic acid amplification / detection reagent, and a sample are added to one container. Then, by applying an external stimulus, the inactivated nucleic acid inactivating reagent is activated. The nucleic acid inactivating reagent thus activated reacts with all contaminating nucleic acids in the container, rendering these contaminating nucleic acids non-reactive with all subsequent steps of the treatment. The nucleic acid inactivating reagent that has not reacted with the contaminating nucleic acid is returned to the inactivated state. The thermophilic proteinase digests contaminating proteins at the optimum temperature for thermophilic proteinase activity, thereby extracting the target nucleic acid. After extraction of the target nucleic acid, the extracted nucleic acid sequence may be amplified using PCR or isothermal amplification, or simultaneously with the amplification, detection by fluorescence is performed by using the isothermal amplification method or the qPCR amplification method. May be.

[Nucleic acid inactivation reagent]
A preferred method of using a nucleic acid inactivating reagent to inactivate contaminating nucleic acids and prevent co-amplification of contaminating nucleic acids is described below. In the present specification, “nucleic acid”, “nucleic acid sequence”, “polynucleotide”, “polynucleotide sequence”, and the corresponding phrases are referred to as single-stranded or double-stranded deoxyribonucleotides, or ribonucleotides having an arbitrary length. Meaning polymers, for example, coding and non-coding sequences of genes, sense and antisense sequences, exons, introns, genomic DNA, cDNA, mRNA precursors, mRNA, rRNA, siRNA, miRNA, tRNA, Ribozymes, recombinant polynucleotides, isolated and purified natural DNA or RNA sequences, synthetic RNA sequences and synthetic DNA sequences, diffusion probes, primers, fragments, gene constructs, vectors, and modified polynucleotides Limited to these It is not. In addition, the terms “nucleic acid”, “oligonucleotide”, and “polynucleotide” in the present specification are not used properly depending on the difference in sequence length, but are used interchangeably.

  In order to prevent the sample from being further contaminated, the nucleic acid inactivation reagent is used in the nucleic acid extraction reaction in which the reaction tube is opened, added to the reaction tube, or removed from the reaction tube as little as possible. And is preferably used in combination with an amplification reaction. Preferably, the nucleic acid inactivating reagent is added together with the extraction reagent and the amplification reagent necessary for amplification and detection of the target nucleic acid in one step performed in the initial stage of the treatment.

  In certain embodiments, chemicals, enzymes, or other biomolecules can be used as the nucleic acid inactivating reagent. Any type of nucleic acid inactivating reagent inactivates the nucleic acid. A contaminating nucleic acid is a nucleic acid present in any of the following. Containers, tubes, plates, or devices used for any process, including nucleic acid preparation / extraction reagents, nucleic acid amplification reagents, nucleic acid use, cause contamination of the target sample, reducing amplification sensitivity and increasing false positive results A plastic container used for any process, including the use of the resulting nucleic acid.

  In a preferred embodiment, the nucleic acid inactivating reagent has the property that it can be present in a sample solution during the nucleic acid extraction and amplification period. As a result, when the container or the reaction tube is opened, an effect of preventing further contamination of the nucleic acid is brought about, and an inactivation process of the nucleic acid can be incorporated into the nucleic acid amplification process. Brought about.

  Preferably, under conditions corresponding to the methods described above, the nucleic acid inactivating reagent has two different states, an inactive form and an active form. Among these, the inactive form is a state in which the nucleic acid is inactivated, and the activated state is a state in which the nucleic acid is inactivated and activation / deactivation of the nucleic acid can be controlled. In certain embodiments, the inactivation reagent may be alternated between an inactive form and an active form, thereby providing a sufficient amount of the inactivation reagent to inactivate contaminating nucleic acids. The inactivated sample that can be added to the sample inactivates the contaminating nucleic acid only in the active form. Thus, after inactivation of the contaminating nucleic acid, the remaining inactivation reagent can be changed to an inactive form so as not to cause further nucleic acid inactivation. This prevents inactivation reagent / contaminating nucleic acid binding from interfering with further reactions during the diffusion amplification process.

  In embodiments of the method where the nucleic acid inactivation reagent retains two different states, the nucleic acid inactivation reagent is switched to the active form prior to extraction and amplification of the target nucleic acid. In a preferred embodiment, the nucleic acid inactivating reagent is non-contaminating after inactivating contaminating nucleic acid against the nucleic acid amplification process and before or simultaneously with incubating the sample with a nucleic acid extraction reagent such as a thermophilic proteinase. Change to active form. The change from the inactive form to the active form, or from the active form to the inactive form occurs through several mechanisms depending on the properties of the specific nucleic acid inactivating reagent. Examples of these mechanisms include the following.

  In another embodiment, the nucleic acid inactivating reagent is in an active form without interfering with a target nucleic acid extraction process, a nucleic acid amplification reagent, or a target nucleic acid amplification process in which a sample is processed with a subsequent thermophilic proteinase. Provide a concentration sufficient to inactivate contaminating nucleic acids.

  Since the nucleic acid fragment or part itself affects the subsequent amplification process, the nucleic acid inactive reagent preferably does not decompose or destroy the contaminating nucleic acid.

[External stimulus]
In certain embodiments, the nucleic acid inactivating reagent switches to the active form only when an external stimulus is applied. It is preferably sufficient to activate the nucleic acid inactivating reagent without substantially activating the nucleic acid inactivating reagent such as a thermophilic proteinase. As used herein, “substantial activation” means, for example, that a thermophilic proteinase is sufficient to be amplified and detected by the above method for a period of time sufficient to convert the nucleic acid inactivating reagent to an active form. It is activated to a degree sufficient to cause extraction of a sufficient amount of nucleic acid.

  In other preferred embodiments, the external stimulus that results in the activation of the nucleic acid inactivating reagent is light with a specific narrow wavelength, white light with a wide range of wavelengths, or ultraviolet light; In a preferred embodiment, the external stimulus is thermal energy. However, the present invention is not limited to this, and the external stimulus may be determined according to the selected nucleic acid inactivating reagent, its active characteristics and inactive characteristics.

  In the preferred embodiment, after the contaminating nucleic acid is inactivated to the nucleic acid amplification process, the nucleic acid inactivating reagent is switched to an inactive form. In some embodiments, removal of the external stimulus switches the nucleic acid inactivating reagent from the active form to the inactive form. In other embodiments, a certain amount of external stimulus is sufficient to convert the nucleic acid inactivating reagent to the active form, and for conversion from the active form to the inactive form, It is necessary to increase the amount of external stimulation. For example, in a preferred embodiment, by applying an external stimulus for a period of time sufficient to convert the nucleic acid inactivating reagent to an active form, the contaminating nucleic acid is changed to an inactive state for the nucleic acid increasing treatment. After that, the nucleic acid inactivating reagent is converted to an active form. In yet another embodiment, the external stimulus converts the nucleic acid inactivating reagent to an active form by causing an irreversible structural change such as photolysis to the nucleic acid inactivating reagent, thereby While sometimes inactivating contaminating nucleic acids, the nucleic acids are not further inactivated after the structural change has occurred. For example, in a preferred embodiment, application of the external stimulus may cause photolysis of the nucleic acid inactivating reagent to convert the nucleic acid inactivating reagent into an active form.

(Inserting agent)
In one embodiment, an intercalating agent such as ethidium, proflavine, or thalidomide that covalently binds to a double-stranded nucleic acid is used as the nucleic acid inactivating reagent. As used herein, “insertion agent” includes a reagent that can be reversibly inserted between two adjacent base pairs in a double-stranded nucleic acid. Examples of well-studied DNA intercalating agents include, but are not limited to, ethidium, proflavine, or thalidomide. In a preferred embodiment, photolysis of the nucleic acid intercalating agent is caused by applying wavelength light having a specific narrow wavelength spectrum, white light having a wide spectrum, or ultraviolet light, and the nucleic acid intercalating agent To the active form.

  In a preferred embodiment, ethidium monoazide (EMA) is used as the intercalating agent. This disclosure is incorporated herein by reference to New Zealand Patent 545,894. The New Zealand Patent 545,894 describes the use of EMA to inactivate contaminating nucleic acids. EMA forms a complex with contaminating nucleic acids. EMA is a photoreactive analog of ethidium bromide, and the amino group at the 8-position in ethidium bromide is substituted with an azide group. Under non-irradiation of white light, EMA is inserted into double-stranded nucleic acid in the same manner as ethidium bromide. However, under light irradiation, EMA is photoactivated and covalently binds to the double-stranded nucleic acid (Bolton and Kearns, 1978; Garland et al., 1980).

  When undergoing photoactivation, the azide group at the 8-position in EMA is photochemically degraded using light having a long wavelength of about 400 nm or longer (Bolton and Kearns, 1978). Photodegraded EMA can be covalently crosslinked to nucleic acids at binding sites via nitrene radicals (Hardwick et al., 1984; Cantell et al., 1979). The remaining unbound nitrene in solution is converted to hydroxylamine (Graves et al., 1981), which reacts with the aldehyde and / or ketone component of the solution, preferably cytosine. Produces an oxime (Freesee et al., 1961). After light irradiation and the resulting photolysis, EMA loses the ability to covalently bind to unbound nucleic acids. Therefore, even when light is irradiated (for example, in a real-time PCR machine), it does not affect the results obtained by subsequent processing. Furthermore, since the covalently crosslinked nucleic acid is not affected in the subsequent nucleic acid amplification step, the interference is removed (Nogva et al., 2003).

  Preferably, EMA is added with a minimal titer sufficient to bind to all exogenous nucleic acids while minimizing the production of free nitrene in solution with the production of hydroxylamine. Since the amount of contaminating nucleic acid in commercially available amplification reagents varies from reagent to reagent, each group of amplification reagents optimizes the EMA titer and ensures maximum removal of contaminating nucleic acids while maintaining amplification sensitivity.

  In a preferred embodiment, the amount of EMA added is an amount sufficient to bind to contaminating nucleic acid. In a preferred embodiment, EMA is also used at a concentration of 1-5 μg / ml. More preferably, the concentration of EMA is about 3 μg / ml.

[Mesophilic nucleic acid modifying enzyme]
In embodiments where the sample cells containing the target nucleic acid for amplification are dead or damaged sample cells, other nucleic acid inactivating reagents are used. The nucleic acid inactivating reagent does not have the ability to diffuse into the cell material. Specifically, a large molecule such as a mesophilic diffusion modifying enzyme is used. Preferably, the enzyme used is one that does not cause the hydrolysis or modification of the oligonucleotide necessary for PCR, so for example, a nuclease that produces a modification or hydrolysis of short single strand diffusion cannot be used. However, in some embodiments, if the oligonucleotide used in the amplification step is modified to be resistant to nuclease cleavage, short single-stranded nucleic acids are modified or hydrolyzed. A nuclease to be used may be used. For example, a 5 ′ modification of a single stranded oligonucleotide is utilized to render the oligonucleotide resistant to a 5 ′ specific exonuclease.

  In a preferred embodiment, the nucleic acid modifying enzyme is used in combination with a nucleic acid extraction enzyme. As such a nucleic acid extraction enzyme, a nucleic acid extraction reagent having a temperature activity characteristic different from that of the intermediate temperature nucleic acid modifying enzyme, for example, a thermophilic proteinase is used. In the above embodiment, the activation of the intermediate temperature nucleic acid modifying enzyme is applied with temperature energy, and the temperature of the intermediate temperature diffusion modifying enzyme is suitable for the activity, but the temperature does not reach the activation of the thermophilic proteinase, Increase the temperature of the intermediate temperature nucleic acid modifying enzyme. For example, in a preferred embodiment, the mesophilic nucleic acid modifying enzyme is incubated at 20 ° C. to 45 ° C. for a time sufficient to convert the mesophilic nucleic acid modifying enzyme from an inactive form to an active form. In another preferred embodiment, the mesophilic nucleic acid modifying enzyme is incubated at 65 ° C. to 80 ° C. for a time sufficient to convert the mesophilic nucleic acid modifying enzyme from an inactive form to an active form.

  In some embodiments, mesophilic nucleic acid modifying enzymes such as nucleases are used, particularly in combination with PCR, qPCR, or RT-PCR amplification methods.

  In other embodiments, the mesophilic nucleic acid modifying enzyme comprises a double strand specific endonuclease (eg, a restriction endonuclease); a double strand specific exonuclease; a single strand specific nuclease with low activity against short molecules; It may be a nucleic acid modifying enzyme specific for a strand nucleic acid.

  In certain embodiments, the intermediate temperature nucleic acid modifying enzyme may be T7 exonuclease, T7 endonuclease 1, ExoIII, a frequently cut restriction endonuclease, or any mixture of the above.

  In other embodiments, mesophilic nucleic acid modifying enzymes are used to inactivate contaminating nucleic acids associated with reverse transcription PCR. The intermediate temperature nucleic acid modifying enzyme may be any of RNAse, RNAseH, or RNA modifying enzyme.

  After the sample is treated with a nucleic acid inactivating reagent and the nucleic acid inactivating reagent is at a suitable level or deactivated, the target nucleic acid can be extracted from the sample. A method for extracting a target nucleic acid using a thermophilic proteinase is described below.

[Nucleic acid extraction]
A suitable method for extracting a target nucleic acid from a biological sample containing cells will be described below. The phrases “extract” and “extract” in this specification refer to a process that increases the availability of the nucleic acid contained in a sample in other processes. What is suggested by the concept of nucleic acid extraction is that the target nucleic acid is sufficiently free from interfering substances such as inhibitors, interfering substances, nucleases, other enzymes, and nucleoproteins, and is also effective in other operating methods It is to be. In the above-described apparatus, there is no point in purifying the nucleic acid from a non-interfering substance, and the nucleic acid does not necessarily have to be purified from a non-interfering substance. The nucleic acid treatment minimizes the harmful effects of the interfering substances.

  In order to minimize contamination of the sample with contaminated nucleic acids, the above nucleic acid extraction should be performed in conjunction with an amplification reaction in which the reaction tube is opened, added to the reaction tube, and removed from the reaction tube as few times as possible. preferable. Preferably, the extraction is performed in the same reaction tube as the increase in the target nucleic acid. In addition, about US Patent 7,547,510, the content of an indication is used by referring. The above-mentioned US Pat. No. 7,547,510 discloses a nucleic acid extraction method that uses a thermophilic proteinase that can be used in combination with a nucleic acid amplification process in one reaction tube.

〔sample〕
Samples can be obtained from a wide range of substrates including clinical samples, food and beverage samples, or environmental samples. Specifically, the microbial sample is obtained from environmental factors, and in the case of food testing, it is obtained by taking a liquid or solid sample or wiping the solid surface. Traditionally, clinical samples have been collected from tissue, blood, serum, plasma, cerebrospinal fluid, urine, excrement, semen, swab, or saliva. Tissue samples are taken by standard methods such as cell scraping to obtain animal tissue or by biopsy methods. Similarly, blood sampling is routinely performed, for example for pathogen testing, and methods for collecting blood samples are well known in the art. Biological sample retention and processing methods are also well known in the art. For example, the tissue sample may be stored frozen until examination is performed. Furthermore, those skilled in the art will understand that some test samples can be analyzed more easily through fractionation or purification. For example, blood is classified as a serum or plasma component.

  Specific examples of forensic samples include blood, saliva, semen, skin, hair, bone, or teeth. In particular, samples with low levels of target nucleic acids or “low copy number” problems are time-lapse stains, bones, teeth, and hair.

  Samples for species identification, samples for anthropology, samples for phylogenetic analysis, and barcodes are collected from a variety of sources and include archaeological excavations, bones, feathers, hair, and stored in museums. Samples stored in formalin, alcohol, or paraffin wax are also included.

  In a preferred embodiment, the sample containing cells from which the target nucleic acid is extracted is blood, urine, saliva, semen, feces, tissue, wipes, tears, bones, teeth, hair, or muscles. In other preferred embodiments, the sample is a bacterium, fungus, archaea, eukaryote, protozoan, or virus. In another embodiment, the sample is a mixture of seawater, fresh water, ice, soil, waste, and food. In other preferred embodiments, the sample is blood, urine, saliva, semen, feces, tissue, wipes, bone, teeth, hair, or mucus. The sample may be skin, feathers, or a preserved or historical sample.

[Thermophilic proteinase]
In a preferred embodiment, a thermophilic proteinase is used to extract nucleic acids from a biological sample. Thermophilic proteinases exhibit proteolytic activity at high temperatures. “Thermophilic proteinase” refers to a proteinase that exhibits optimal proteolytic activity at a high temperature of, for example, about 65 ° C. to 80 ° C., and has been isolated from a thermophilic organism having optimal proteolytic activity as described above. Proteinases isolated from organisms other than proteinases and thermophilic organisms are included. Such proteinases include proteinase variants, including modified or genetically engineered variants.

  A thermophilic proteinase that is particularly suitable for use in the method of the present embodiment is a thermophilic proteinase that can be heat denatured at about 90 ° C. and / or a thermophilic proteinase that produces autocatalysis, and preferably, for example, about 90 ° It is a thermophilic proteinase that is permanently heat-inactivated at the above high temperatures. In certain embodiments, preferred features of the thermophilic proteinase used include the following:

1) substantially stabilized and activated in the range of about 65 ° C to 80 ° C;
2) easily deactivated and / or denatured above about 90 ° C.
3) The activity is suitably reduced below 40 ° C., and for example, it has a temperature-activity characteristic such that the mesophilic enzyme for removing the cell wall present together is not degraded.

  A preferable incubation temperature required for activating the thermophilic proteinase and extracting the target nucleic acid through cell degradation, protein digestion, or cell wall digestion is 75 ° C. The preferred incubation temperature required to generate the thermophilic proteinase autocatalyst is 95 ° C. The preferable temperature is merely an example, and is not limited to these. The proteinases also have different properties for both enzyme activity and stability over a temperature range, and the kinetics of such enzymes are known to those skilled in the art. Furthermore, the kinetics of the enzyme with respect to such a proteinase is confirmed by the minimum necessary experiments.

  In a preferred embodiment, the thermophilic proteinase is an EA1 proteinase, a neutral proteinase isolated from Bacillus sp. Strain EA1, and variants thereof, and an Ak1 proteinase, a serine protease isolated from Bacillus sp. Strain Ak1, And variants thereof.

  In a particularly preferred embodiment, EA1 proteinase is used. The EA1 proteinase is activated and degraded (by autolysis) with changes in temperature. For example, when incubated at 65 ° C. to 80 ° C., EA1 proteinase is in an active form. Under this temperature, cells in the sample are degraded, and the EA1 proteinase degrades contaminating proteins, and rapidly removes the DNA-degrading nuclease at a temperature at which the DNA-degrading nuclease is in an inactive form. Thereby, degradation of the target nucleic acid is suppressed to a minimum.

In certain embodiments, the extraction of the target nucleic acid from the sample in a closed system comprises
1) adding at least one thermophilic proteinase to a sample containing nucleic acid for testing;
2) incubating the sample at 65-80 ° C. for a preferred period required to generate one or more of cell degradation, protein digestion, cell wall enzyme digestion, and the thermophilic proteinase Is stable and active at about 65-80 ° C., while it is inactivated and / or denatured when the sample is incubated at 90 ° C. or higher without the need for further addition of denaturing enzymes.

  In a preferred embodiment, a thermophilic proteinase is used, but the enzyme used with the above method is not limited thereto.

[Other extractive enzymes]
In certain embodiments, the mesophilic enzyme exhibits an active form at a temperature lower than that of the thermophilic proteinase and is mixed with the thermophilic proteinase to activate the thermophilic proteinase to extract nucleic acids within the closed system. Before proceeding, it is used to weaken and / or remove the cell walls of plants, mycelium, bacteria, spores, biofilms. The practice of the method is based on the proteinase and / or proteinase / cell wall degrading enzyme that exhibits different activities at different temperatures. By repeatedly setting different temperatures, different enzyme activities can be produced without opening the system and adding new reagents.

In a preferred embodiment, the method using a mesophilic enzyme and a thermophilic proteinase further comprises:
adding at least one mesophilic enzyme and at least one optional thermophilic enzyme to a sample containing a target nucleic acid for testing;
b incubating the sample for less than about 40 ° C. for a preferred period necessary to remove the cell walls of the sample cells.

  The preferred initial incubation temperature required to cause cell wall removal due to the activity of the mesophilic enzyme is 37 ° C. Again, the initial incubation temperature is an example and is not limited to this.

  In certain embodiments, the mesophilic enzyme may be cellulose or lysozyme.

  The thermophilic proteinase may be used in combination with other hydrolases that cause cell wall destruction or weakening. This allows the treatment of subjects, including plant cells, rugged bacterial cells, or fungal spores, obtained from a subject, including the environment (eg, soil, stone, water, plant material samples) or tissue, or fluid from the subject. Target nucleic acid can be extracted from difficult cells.

  The sample is treated with a nucleic acid inactivating reagent, treated with a reagent to extract nucleic acid, and the target nucleic acid contained in the sample is amplified after the extraction reagent is deactivated. The A known nucleic acid sequence of interest is amplified by a PCR-type method or an isothermal method.

[Amplification of target nucleic acid]
A preferred nucleic acid amplification method will be described below. As the method, a sample is treated with a nucleic acid inactivating reagent so as to suppress contaminated nucleic acid, and after extracting the target nucleic acid from the sample by any of the methods, the region of interest of the sample A PCR-type nucleic acid amplification method and an isothermal nucleic acid amplification method for amplifying the target nucleic acid. In certain embodiments, the target nucleic acid amplification process may be automated.

[PCR amplification]
PCR reagents specifically include a set of primers corresponding to each target nucleic acid, a DNA polymerase (preferably a thermostable DNA polymerase), a DNA polymerase cofactor, one or more deoxyribonucleotides Includes 5'-triphosphates (dNTP's) or similar nucleosides. Other optional reagents and materials used for PCR are described below.

  DNA polymerase is an enzyme that adds a deoxynucleoside monophosphate molecule to the end of a primer (usually the end on the 3 ′ hydroxyl group side) in a primer / template complex, and this deoxynucleoside monophosphate molecule is a template. It is added in a format dependent on. The synthesis of the extension product proceeds from the 5 'end to the 3' end of the newly synthesized strand until the synthesis is complete. Useful DNA polymerases include, for example, Taq polymerase, E.I. E. coli DNA polymerase I, T4 DNA polymerase, Klenow polymerase, reverse transcriptase, and others known in the art. Preferably, the DNA polymerase is thermostable, i.e. stable to heat, and is selectively in active form at elevated temperatures, particularly at temperatures used in priming and stretching of DNA strands. More specifically, the thermostable DNA polymerase does not substantially become an inactive form at the high temperatures utilized in the polymerase chain reaction. Such temperatures will vary depending on the number of reaction conditions including pH value, nucleotide composition, primer length, salt concentration, and other conditions known in the art.

Particularly useful polymerases are those derived from various Thermus bacterial species, which include Thermus aquaticus, Thermus thermophilus, Thermus filiformis, and Thermus flavus. Other useful thermostable polymerases are obtained from a variety of microbial sources such as Thermococcus literalis, Pyrococcus furiosus, Thermotoga sp, and International Publication No. WO-A-89 / 06691 (publication date 1989). July 27, 2007). Some of the useful thermostable polymerases are such as AmpliTaq ^™ , Tth, and UlTma ^™ (Perkin Elmer), Pfu (Stratagene), Vent and Deep-Vent (England Biolabs). Are commercially available. Other polymerases are complexed with other molecules that render the polymerase in an inactive form until high temperatures are applied. Specifically, an antibody is used. An example of such a polymerase is Platinum (registered trademark) Taq (manufactured by Invitrogen). Many techniques for isolating naturally-occurring polymerases obtained from organisms and techniques for producing enzymes synthesized by genetic engineering using gene recombination techniques are known.

  DNA polymerase cofactor refers to a non-protein compound that determines enzyme activity. Thus, in the absence of cofactors, the enzyme is in catalytically inactive form. Numerous materials are known as cofactors including, but not limited to, manganese and magnesium salts such as chlorides, sulfates, acetates, fatty acid salts and the like. Magnesium chloride and magnesium sulfate are preferred.

  Further, two or more deoxyribonucleotide-5′-triphosphates are required for PCR, and the two or more deoxyribonucleotide-5′-triphosphates are any of dATP, dCTP, dGTP, and dTTP. Two or more are mentioned. Analogs such as dITP, dUTP, and 7-deaza-dGTP are also useful. Preferably, four common triphosphates (dATP, dCTP, dGTP, dTTP) are used together.

  The PCR reagent is used at a concentration suitable for causing amplification of the target nucleic acid in the PCR. The minimum amount of primer, DNA polymerase, cofactor, deoxyribonucleotide-5'-triphosphate required for amplification is well known, and their preferred ranges are also well known. The minimum amount of DNA polymerase is at least about 0.5 units / 100 μl of solution, preferably about 2-25 units / 100 μl, more preferably about 7-20 units / 100 μl. . Other amounts are useful in a given amplification system. “Unit” is defined as the amount of enzyme activity required to incorporate 10 nmol of all nucleotides (dNTP's) into an extended nucleic acid strand at 74 ° C. for 30 minutes. The minimum amount of primer is at least about 0.075 μmol, preferably 0.1-2 μmol, although other amounts are well known in the art. The cofactor is usually present in an amount of 2-15 mmol. The amount of each dNTP is usually about 0.25 mmol to 3.5 mmol.

  The PCR reagents may be supplied independently, may be supplied in various combinations, or may be supplied by a buffer having a pH value of 7-9. The buffer solution in this case may be any suitable buffer solution, many of which are known in the art.

  Examples of other reagents used for PCR include antibodies specific for thermostable DNA polymerase. The antibody is used to suppress the action of the polymerase before the amplification treatment. Preferably, the antibody is specific for a thermostable DNA polymerase, inhibits the enzymatic activity of the DNA polymerase at a temperature of less than about 50 ° C., and is inactivated at an elevated temperature. Useful antibodies include monoclonal antibodies, polyclonal antibodies, and antibody fragments. Preferably, the antibody is monoclonal. Antibodies are described in Harlow et al. , Antibodies: A Laboratory Manual, Cold Spring Harbor, N .; Y. (1988) may be used to adjust using known methods.

  After supplying the PCR reagent, temperature cycling is realized using a heater and a controller. PCR reagents may be multiplexed so that a plurality of target nucleic acids can be analyzed simultaneously.

  In a specific embodiment, a general PCR method is employed. In other particularly preferred embodiments, qPCR, reverse transcription PCR, or multiplex PCR methods are employed.

“QPCR”, “quantitative PCR”, and “real-time PCR” are used interchangeably as referring to a polymerase chain reaction (PCR) method that simultaneously amplifies and quantifies a target nucleic acid. In qPCR analysis, a positive reaction is detected by the accumulation of a fluorescent signal. C _T (cycle threshold) is defined as the number of cycles required for the fluorescent signal to exceed the threshold (exceeding the background level). The _CT level is half proportional to the amount of the target nucleic acid in the sample (the lower the _CT level, the more target nucleic acid in the sample).

[Isothermal amplification method]
Another aspect of the present disclosure is an isothermal amplification method for detecting a target nucleic acid, which is a method based on target nucleic acid-dependent amplification of a signal from a detectable label bound to a nucleic acid probe. is there. The isothermal amplification method is disclosed in PCT application PCT / NZ2007 / 000197. The isothermal amplification method can be performed by strand displacement amplification method, rolling circle amplification method, loop-mediated isothermal amplification method, chimeric primer-derived nucleic acid isothermal amplification method, Q-beta amplification system, or OneCutEventAmplificatioN.

  Techniques applied during the isothermal amplification process are nuclease chain reaction (NCR) and RNAse-mediated nuclease chain reaction (RNCR). Both replace strand displacement with selective degradation of one of the DNA strands. If the one DNA strand contains ribonucleotides, the treatment may be initiated by a restriction nuclease or RNAseH. In the polymerase nuclease chain reaction (PNCR), after nuclease cleavage in the presence of target DNA, extension treatment using DNA polymerase and RNAse-mediated detection (RMD), which is a strand degradation method using RNAseH for DNA: RNA hybrids, are performed. . RMD is an effective linear amplification system that is often used in combination with other methods. Tandem repeat restriction enzyme-promoted (TR-REF) chain reaction or inverted reverse complement restriction enzyme-promoted (IRC-REF) chain reaction is based on two methods based on the periodic generation of detection probes containing tandem repeats. It is. These repeats are replicated by DNA polymerase when a particular oligonucleotide trigger acts as a primer. The restriction endonuclease then attacks the newly formed double stranded DNA, thereby releasing the original primer and the secondary primer and starting two new cycles. Isothermal amplification reactions can be multiplexed to simultaneously assay multiple target nucleic acids of interest.

  In addition, there is a nucleic acid having strand invasion properties, and by such strand invasion, the target sequence is not initially single-stranded, but the complementary strand of the target nucleic acid is replaced and the double strand of the target probe is formed. Alternatively, it results in the formation of a triplet of the target probe. Peptide nucleic acids (PNAs) and their derivatives are capable of strand invasion, which includes the target nucleic acid binding region containing PNAs, and the currently disclosed probes are completely altered to single stranded. Used to detect non-nucleic acid. The use of a target binding region comprising PNAs is envisioned, particularly in circular probes, where the target binding region of the probe may be substantially double stranded prior to formation of the target probe hybrid.

  A “target binding domain” and its corresponding “target binding region” are present in a nucleic acid molecule and cause hybridization between the target binding region and the target nucleic acid to a nucleic acid sequence present in the target nucleic acid. Refers to nucleic acid sequences that are sufficiently complementary to form a target probe hybrid.

[SNP analysis]
There are two growing fields where DNA-based diagnostics are applied: tailored medicine using single nucleotide polymorphism (SNP) analysis, and pedigree analysis of livestock. An indication of the importance of these areas is the International Hapmap Project, which aims to create a complete halotype map of the human genome. Single nucleotide polymorphisms provide markers for specific characteristics or diseases. Numerous platforms and techniques are available to identify alleles at established SNP loci. Single nucleotide polymorphisms can be confirmed by various polymerase reference techniques such as PCR and primer extension. In some cases, the product of the primer extension system is analyzed in a multiplex using mass spectrometry. Other methods, such as invasion techniques, use enzymes that are sensitive to DNA sequence mismatches (Oliver, 2005). Alternatively, ligase-based analysis and nuclease-based analysis, or methods based on the hybridization dynamics of short oligonucleotides to the polymorphic region of the genome may be used. An example of the latter is the Affymetrix high density array.

[Amplification detection]
In certain embodiments, the method for amplifying a target nucleic acid is based on the detection or measurement of a signal from a label, wherein the signal is preferably the luminescence of a probe labeled with a luminescent label. “Label” refers to a component that can be attached to any atom, molecule, compound, or nucleic acid that provides a detectable signal or interacts with a secondary label to remove the secondary label from the secondary label. Used to modify the detectable signal emitted. Preferred labels are luminescent compounds that produce a detectable signal generated by fluorescence, chemiluminescence, or bioluminescence. Further preferred labels are luminescent compounds that reduce their signal or become undetectable when in close proximity to a masking group (eg, a quenching chromophore). In a particularly preferred embodiment, nucleic acid amplification is detected by fluorescence.

  Luminescent labels may be used in PCR methods and isothermal detection methods. The mechanism by which the luminescence of a compound is attenuated by a second compound is described in Morrison, 1992, in Nonisotopic DNA Probe Techniques (Kricka ed., Academic Press, Inc. San Diego, Calif.), Chapter 13. This mechanism includes fluorescence energy transfer (FRET), non-radioactive energy transfer, long range energy transfer, dipole-coupled energy transfer, and Forster energy transfer. The main requirement for FRET is that the emission spectrum of one of the compounds (the energy donor) must overlap the absorption spectrum of the other compound (energy acceptor). Styer and Haugland, 1967, Proc. Natl. Acad. Sci. U. S. A. 98: 719 (incorporated herein by reference) shows that the energy transfer efficiency of some common emitter-quencher pairs can approach 100% when the separation distance is less than 10 mm. The energy transfer rate decreases in proportion to the sixth power of the distance between the energy donor molecule and the energy acceptor molecule. In conclusion, a small increase in separation distance greatly reduces the energy transfer rate, increasing the fluorescence of the energy donor, and reducing the energy acceptor when the quencher chromophore is also a fluorophore. Produced fluorescence. In the method, signal emission of a label (preferably a fluorescent label) bound to the probe is detected.

  The exposure of the detection sequence means that the detection sequence can be collected for detection purposes, for example, it can be collected for binding to a detection probe. Conversely, "hidden", "shielded", and grammatical equivalents mean that the elements described by these phrases are not harvestable. For example, when the detection sequence is bound to a nucleic acid molecule other than the detection probe, it is hidden or shielded. “Hybridization” and the grammatically equivalent phrase are the formation of multiple bond structures, such as double bond structures, by complementary base pairing of two or more single stranded nucleic acids. To do.

  It should be noted that a labeling system that indicates cleavage of the label from components that can bind to the solid matrix may be used. As an example of this, there is a biotin label capable of binding to immobilized avidin, and when the probe is not cleaved, a secondary label present at the opposite end of the probe can be bound. Such a method is used for detection using a dipstick. However, many detection systems use labels that are distinguishable by nanopore technology. A plurality of labels can be used as the label, but the method described in this specification can be applied to the detection of a probe labeled with one label. A cleaved probe is detected when the label (eg, fluorophore) is sufficiently separated from the masking group (eg, quencher) by cleavage or a probe denaturation process by cleavage. This reduces the interaction between the masking group and the label, allowing the signal to be released. The “masking group” is any atom, molecular compound, or component capable of interacting with the label and reducing signal emission from the label. Separation of the label and the masking group by a cleavage or probe denaturation process in turn increases the detectable release of signal from the attached label. Depending on the label, signal emission includes luminescence, particle emission, appearance or disappearance of colored compounds, and the like.

  In the following, preferred luminescent labels and masking groups that interact to change the luminescence from the label are described. The “chromophore” refers to a non-radioactive compound that absorbs energy as light. Some luminescent populations emit light by a chemical reaction when excited and cause chemiluminescence, while others emit light by absorbing light and emit fluorescence when excited. “Fluorophore” refers to a compound capable of emitting fluorescence (that is, absorbing light of a certain frequency and emitting light of another frequency that is usually lower than the above frequency).

  “Bioluminescence” refers to one form of chemiluminescence, and the luminescent compound in the bioluminescence is a compound that exists in the living body. Examples of compounds that perform bioluminescence include bacterial luciferase and firefly luciferase. Also, “quenching” refers to a decrease by the second compound in the fluorescence emitted by the first compound, regardless of the mechanism. Specifically, in the quenching, the first compound and the second compound are required to be close to each other. As described herein, it can be said that either the compound is quenched or the fluorescence of the compound is quenched, and these expressions explain the same phenomenon.

  Many fluorophores and chromophores disclosed in the field of the present invention can be applied to the method of the present invention. A preferred fluorophore and quenching chromophore pair is such that the emission spectrum of the fluorophore and the absorption spectrum of the chromophore overlap. Preferably, the fluorophore has a large Stokes mutation (a large difference between the wavelength at the maximum absorption and the wavelength at the maximum emission) so as to minimize the interference due to the scattered excitation light.

Suitable labels well known in the art include fluorescein and derivatives (eg, FAM, HEX, TET, and JOE); rhodamine and derivatives (eg, Texas Red, ROX, and TAMRA); lucifer yellow, and coumarin derivatives (eg, , 7-Me ₂ N-coumarin-4-acetate, 7-OH-4-CH 3 - coumarin-3-acetate, and _{7-NH 2 -4-CH 3} - coumarin-3-acetate (AMCA)) include However, it is not limited to these. FAM, HEX, TET, JOE, ROX, and TAMRA are sold by Perkin Elmer, Applied Biosystems Division (Foster City, Calif.). Texas Red and many other suitable compounds are sold by Molecular Probes (Eugene, Oreg.). Examples of chemiluminescent and bioluminescent compounds that may be suitable for use as the energy donor include luminol (aminophthalhydrazide) and derivatives, and luciferase.

  In most embodiments, it is preferred that the detectable label is a luminescent label and the masking group is a quencher (eg, a quenching chromophore), while other detectable labels and masking groups are also present. ,Conceivable. For example, the label may be an enzyme, and the masking group may be an inhibitor of the enzyme. If the enzyme and inhibitor are present in close proximity to interact, the inhibitor should also inhibit the activity of the enzyme. Upon cleavage or denaturation of the probe, the enzyme and inhibitor are separated and can no longer interact as the enzyme is active. A wide variety of enzymes that can catalyze reactions that produce detectable products, and inhibitors of the activity of such enzymes, are well known to those of skill in the art (eg, β-galactosidase and horseradish peroxidase).

[Method of combining inactivation of contaminating nucleic acid with extraction and amplification of target nucleic acid]
The method for inactivating contaminating nucleic acid, the method for extracting the target nucleic acid from the sample, and the method for amplifying the extracted nucleic acid are compatible with each other, and may be combined in one closed system. In a preferred embodiment of the invention, the closed system comprises a single container or tube.

  Preferably, the reagent that inactivates the contaminated nucleic acid, the reagent that extracts the target nucleic acid from the sample, and the reagent that amplifies the extracted nucleic acid open the closed system after the inactivation of the contaminated nucleic acid. It is added in such a way that it can be dispensed with.

  In certain embodiments of the invention, reagents that are compatible with each other are supplied as a mixed mixture and the system is closed after supply. In other embodiments of the invention, the reagents are incompatible with each other, so that these reagents are functionally isolated from each other and supplied in sequence.

  In a particularly preferred embodiment, the nucleic acid inactivating reagent is compatible with the nucleic acid extraction reagent (eg, thermophilic proteinase), and the nucleic acid inactivating reagent and the nucleic acid extraction reagent are mixed with each other. Can be supplied as a single mixture. For example, a mesophilic nucleic acid modified inactivation reagent exhibits an active form at about 25 ° C to 40 ° C and an inactive form at about 65 ° C to 80 ° C. On the other hand, the thermophilic proteinase exhibits an inactive form at about 25 ° C to 40 ° C and an active form at about 65 ° C to 80 ° C. The above is merely an example, and the present invention is not limited to this. In another preferred embodiment, the inactivation reagent and the nucleic acid extraction reagent are further compatible with a nucleic acid amplification reagent, and these reagents can be supplied as a mixture. As an example, the nucleic acid amplification reagent is not affected by any of the nucleic acid inactivation reagent and the nucleic acid extraction reagent and the inactivation and extraction of the nucleic acid generated by them. For example, the DNA polymerase is resistant to proteolytic cleavage by a thermophilic proteinase.

  Under such circumstances, the nucleic acid inactivating reagent is supplied in combination with the nucleic acid extraction reagent and the nucleic acid amplification reagent. For example, the nucleic acid inactivating reagent includes all components (buffer solution containing deoxyribonucleotides, divalent ions, oligonucleotide primers, and DNA polymerase) necessary for generating a PCR reaction other than the target nucleic acid to be amplified. May be supplied with a basic mixture of PCR.

  In other embodiments, where the nucleic acid amplification reagent is not fully compatible with other reagents, the reagents may be functionally isolated from one another and supplied in sequence in a closed system. For example, some DNA polymerases, such as Taq DNA polymerase, are degraded by the thermophilic proteinase in the nucleic acid extraction reagent, so it is necessary to consider means for supplying the DNA polymerase after nucleic acid extraction. Possible supply means include the following four. (1) Means for supplying the DNA polymerase and other sensitive reagents after the nucleic acid inactivation treatment and extraction treatment. In this supply means, the reagent is supplied through the inlet using a microfluidic or solid dispenser. (2) Supply means for adding the DNA polymerase and other sensitive reagents to the nucleic acid inactivating reagent and the nucleic acid extraction reagent in a protected form. The supply means is supplied with a bead or film in which the other sensitive reagent is encapsulated. (3) The DNA polymerase is modified so that the DNA polymerase is protected from the thermophilic proteinase, for example, by adding an antibody. (4) Use a novel polymerase that is resistant to proteolytic cleavage. In certain embodiments, the nucleic acid amplification reagent may be supplied using a microfluidic or solid dispenser. In another embodiment, the nucleic acid amplification reagent is added using a microcapsule containing the nucleic acid amplification reagent.

  In a preferred embodiment, the microcapsules are pre-supplied in the container or tube in which the nucleic acid is amplified. In another preferred embodiment, the microcapsule is a non-heat resistant capsule. In still another preferred embodiment, the non-heat-resistant microcapsules are agarose or wax material beads. In yet another preferred embodiment, the non-heat resistant microcapsules release the amplified detection reagent when exposed to a temperature sufficient to dissolve or degrade the non-heat resistant microcapsules.

  In certain embodiments where the nucleic acid inactivation reagent is used in combination with a quantitative PCR amplification method, it should be considered to use an EMA fluorophore as a quencher and / or reporter dye for use with quantitative PCR. is there.

[Device for inactivation, extraction and amplification of contaminating nucleic acids]
In a preferred embodiment, the closed system comprising a single container or tube for inactivation, extraction and amplification of contaminating nucleic acids may be realized by a device. FIG. 2 shows a preferred embodiment of the apparatus. The preferred device comprises an external stimulus source that changes the nucleic acid from an inactive form to an active form. For example, in certain embodiments, a light source of wavelength light having a specific narrow spectrum, a white light source having a broad spectrum, or a light source of UV light causes photolysis and activates the EMA. Since the above apparatus enables a reaction in a closed system, a degree of physical adjustment is required for the reaction that occurs during the period from the introduction of a sample into the closed system until the result is obtained. Preferably, light and temperature are utilized to start and stop the continuous chemical reaction so that multiple stages of processing can be performed without the use of complex pumps, valves or fluids. Light and heat can be controlled by a simple device with microelectronics, LEDs, Peltier plates, or incandescent bulbs.

  In a preferred embodiment, the apparatus corresponds to the reaction conditions of each stage for all stages of the combination method from inactivation of contaminating nucleic acids to extraction of target nucleic acids from samples and amplification of target nucleic acids. This system can be incorporated into existing technology.

  In a preferred embodiment, the device comprises a single chamber. In other embodiments, the chamber is dark or shielded. In yet another embodiment, the chamber holds an external tube (eg, a PCR tube) or an external plate (eg, a 96 well microtiter plate) provided within the device. In yet another embodiment, the apparatus comprises an inlet, an outlet, a chamber, a detector of the emitted fluorophore, and an excitation light source. In a preferred embodiment, the apparatus further comprises means for controlling the temperature in the chamber. In some embodiments, the device also includes a light source that activates the nucleic acid inactivating reagent. In a particular embodiment, the light source that activates the nucleic acid inactivating reagent can be an incandescent lamp, a fluorescent lamp, or a light emitting diode array.

  In other embodiments, the device further comprises microfluidics, a microchip, nanopore technology, and a miniature device. The device or the components of the device may be disposable. The device may also be a handheld device.

  The apparatus and method of the present invention provides a wide range of nucleic acid diagnostic techniques in which purification of nucleic acids to remove contaminants is particularly beneficial, or the use of the apparatus and method of the present invention to achieve similar beneficial results. It can be applied to possible diagnostic techniques.

[Computer-related embodiments]
Device control can be achieved by standard electronic methods using hardware, software, and firmware specific to the thermal cycling device. Similarly, an integrated detection system can use similar programmable equipment.

  The data obtained from the detector is diverse, and when the detection device is used to detect a specific substance, a simple Yes / No format indicating whether the specific substance has been detected or not. It may be data. Moreover, since the data may be real-time data obtained by measuring the time when the signal reaches a predetermined threshold value, quantitative data can be provided. Similarly, the electrophoresis data may be such that the fluorescence peak value reaches a detector provided at a position along the capillary electrophoresis apparatus.

  Data analysis can be performed using an external electronic port, wireless technology, an internal storage device, or a computer program supplied to the device via internal firmware. As an example, in the case of an apparatus having a function of determining the presence or absence of one target nucleic acid, the determination result may be reported in the form of a visual indicator such as light and / or LCD or LED display.

  If more complex data analysis is required, or if more user input is required for the data, the raw data, processed data, or partially processed data can be It can be transferred to an external computer via a removable storage device or communication cable.

  In a specific embodiment, the determination result may be obtained by applying wireless technology to obtain database information, and is stored in the device and uses database information useful for identifying a target nucleic acid in a sample. It may be a thing. The determination result may be binarized data (for example, presence or absence of target nucleic acid), but may be quantitative data or multivariate data.

〔Example〕
Hereinafter, although an example is shown and an aspect of this indication content is explained, the present invention is not limited to these examples. Accordingly, various modifications and additions can be made within the scope of the following claims.

Example 1: Use of ethidium monoazide in a combined nucleic acid amplification process
〔background〕
The experiments described below show that, in a closed system method that extracts and amplifies a target nucleic acid in a sample, when the contaminating nucleic acid is inactivated by treatment with ethidium monoazide, the amplification detection accuracy is greatly increased unexpectedly. It is shown that.

  The experiment was performed on a dilution series of Escherichia coli cells, and the presence or absence of the cells was detected using a 16S rRNA oligonucleotide universal primer. These primers are typical of the type used for microbial analysis. These primers are advantageous in that they can be applied to the detection of any known bacterial species, and are disadvantageous in that they also detect contaminating bacterial DNA other than the target. The purpose of the above experiment is to demonstrate that it is possible to perform reagent purification, DNA extraction, and DNA amplification by qPCR by controlling the reaction using only light and heat in a single sealed container. As well as demonstrating that the lower limit of detection is greatly improved by the above process.

For example, with 16S rRNA oligonucleotide universal primers, under normal qPCR conditions using more than 25 cycles, there is a higher risk of false positives or misidentifying organisms in the sample. This is illustrated in FIG. The broken line in FIG. 2 (including Incidentally, qPCR reaction, only ultrapure water), showing the mean C _T value of the negative control. The average _CT value is about 25 cycles. When EMA is used, the mean _CT value of the negative control increases to 37 cycles. This makes it possible to perform at least another 10 cycles in qPCR, so that the detection sensitivity can be greatly improved before the background level becomes a problem.

[Materials and methods]
In the following experiments, all operations were performed in a PCR hood installed in an airtight laboratory where a positive pressure was generated by the HEPA filter. All reagents used in the experiment are unopened. Moreover, the tube, PCR plate, and filter used in the experiment are unopened. All material surfaces were disinfected with sodium hypochlorite at a concentration of 1% prior to the experiment.

〔reagent〕
The following reagents were used.
1.1 U / μl of EA1 proteinase (ZyGEM)
2. 2. GIBCO UltraPure ^™ (Invitrogen) distilled water Quanta Bioscience qPCR reagent 4. 0.1 mg / ml ethidium monoazide Optically clear, 96-neck PCR plate (Axygen)
6). Maximum recovery filter tip (Axygen)
7. 10 μM qPCR primer.

The qPCR primer has the following sequence:
1. Forward primer: GTCGTCAGCTCGGTGTGTGA (SEQ ID NO: 1)
2. Reverse primer: GCCCGGGAACGTATTCAC (SEQ ID NO: 2).

The Quanta Bioscience qPCR reagent includes dNTP's, buffer, MgCl ₂ , and Taq DNA polymerase.

〔Method〕
Escherichia coli MG1655 cells were cultured overnight in LB medium. The cells are then centrifuged at 12,000 RCF for 5 minutes and resuspended in water to a cell density of 2 × 10 ⁷ cells / ml. This cell density corresponds to 10 ⁵ cells per ⁵ μl. The cells were serially diluted 1:10 with ultrapure water to make 10 cells per approximately 5 μl. After serial dilution, 5 μl of each dilution was transferred to 8 wells of an optically clear 96 well microtiter plate.

  Under low light conditions, the following cocktail containing EMA was added to 4 replicates of each dilution.

  The following cocktail without EMA was added to the other four replicates of each dilution.

  In addition to the duplicates of each dilution above, four waters were prepared for each reagent cocktail. Since the Taq DNA polymerase was found to be resistant to inactivation by the EA1 proteinase, the EA1 proteinase and the Taq DNA polymerase could be added simultaneously.

  The 96-well microtiter plate was sealed using a clear and adhesive lid and placed in the dark at 4 ° C. for 5 minutes. The 96-well microtiter plate was irradiated with light from a halogen lamp provided at a position 200 mm away and emitting light at 600 W through the lid for 5 minutes while being kept at 4 ° C. Thereafter, the sample was transferred to an Applied Biosystems 7300 real-time PCR system, and the following cycle was performed.

  The PCR step was repeated, and fluorescence was measured by excitation and emission of SYBR (registered trademark) Green (respective wavelengths were 494 nm and 521 nm) in the step of 72 ° C. and 30 seconds.

〔result〕
Figure 3 shows the C _T values obtained when causing qPCR reaction in the absence of EMA. From the results shown in FIG. 3, it can be seen that excitation and detection could be performed in a single container without opening the tube. As is clear from the broken line in FIG. 3, the highest _CT value obtained in this case is about 25 cycles. This is a C _T value by negative control (ultrapure water only). In addition, this _CT value corresponds with about 1,000 cells (genome).

FIG. 4 shows a result obtained by performing the EMA process, which is improved from the result shown in FIG. Note that the EMA treatment is incorporated in all reactions. Here, C _T value of the negative control is increased up to about 37 cycles. Since each cycle indicates that the DNA is doubled, in this case, the detection accuracy is improved about 4,000 times. Adding EMA to the system allows detection of DNA in a sample containing less than 10 cells per reaction.

[Discussion]
From the results of the above experiments, it was found that all steps of the combined treatment including inactivation of contaminating nucleic acids, extraction of target DNA, and PCR amplification of target DNA can be performed in a single sealed container. . All reagents are processed simultaneously, similar to the reaction vessel in which they are stored. The above experimental results show an unexpectedly great effect of inactivating contaminating nucleic acids in a closed system using EMA. As shown in FIG. 4, when EMA was added to the negative control, it was possible to increase the number of amplification cycles to about 37 before the contaminating nucleic acid was detected. Since the reagents required for the decontamination process, extraction process, and detection process are not compatible with each other, it is not expected that all the steps in the above process can be combined and performed in a single closed tube procedure. It was. In this experiment, when the above treatment is combined into one cocktail that is controlled only by external stimuli, the background is greatly reduced, and at the same time, the extraction treatment and the amplification treatment are not suppressed. Could be done. Each step of the treatment is not inhibited even in the presence of a reagent used in other steps. The combined effect of the above is to reduce background contamination, which is a disturbing factor, to the extent that the number of cycles of qPCR amplification is increased by 12 cycles, thereby improving amplification detection accuracy by about 4,000 times.

  One advantage of this new method is that the detection level is improved by more than three orders of magnitude by combining the contaminating nucleic acid inactivation step with the extraction step and the amplification step, compared to the case where such a combination is not performed. I have done it before. Since this improvement in detection level is realized by reducing the background noise value, more amplification cycles can be used. Maintaining a closed system throughout all stages of the reaction (including the inactivation step) also means a significant improvement in preventing contamination of the sample.

  Furthermore, by integrating the inactivation step into the reaction, it becomes possible to treat all reagents and the reaction vessel at the same time. This simplifies the procedure and allows easy automation of the process. In addition, by combining the inactivation step with the extraction step in this way, it becomes possible to detect DNA from living cells, and from contamination by contaminated reagents, sample matrices, dead cells, or plastic containers. It is also possible to prevent DNA.

  In addition, the possibility of false positive diagnosis and inaccurate diagnosis is greatly reduced. Furthermore, since the background noise is reduced as described above, the possibility of identifying a minute and rare sample is greatly increased.

[Example 2: Comparison of EMA treatment, nucleic acid extraction, and nucleic acid amplification combined with EMA pretreatment]
The combination nucleic acid amplification process incorporating the EMA process described in Example 1 was compared with the process of subjecting the sample to EMA pretreatment before extraction and amplification of the target DNA.

  The combined nucleic acid amplification treatment is performed in the same manner as in Example 1.

  The EMA pretreatment method was performed under the following conditions. 0.1 μg / μl of EMA was added to the sample in a 96-well microtiter plate and sealed using a clear, adhesive lid. And it left still in the dark at 4 degreeC for 5 minutes. Thereafter, the sample in the plate in the sealed state as described above was irradiated with halogen light for 5 minutes. This halogen light is emitted at 600 W from a halogen lamp provided at a distance of 200 mm from the plate. In addition, the temperature of the said plate at the time of irradiation was kept at 4 degreeC.

  After the EMA pretreatment, the lid that sealed the plate was removed and Quanta PCR mix, Primer 1, Primer 2, and 1 U / μl of EA1 proteinase were added to each sample. The plate was then resealed using a clear, adhesive lid and the plate was placed in an Applied Biosystems 7300 real-time PCR system. Next, qPCR was performed by the same method as in Example 1.

  The result of the composite EMA method was compared with the result of the EMA pretreatment method, and the difference in amplification detection sensitivity between these methods was quantified. As a result, it was found that the amplification detection sensitivity of the composite EMA method is increased as compared with the detection sensitivity of the EMA pretreatment.

[Example 3: Use of mesophilic nucleic acid modifying enzyme in complex nucleic acid amplification treatment]
A mesophilic nuclease that inactivates contaminating nucleic acid, and a thermophilic proteinase that exhibits an inactive form in the presence of DNA polymerase and has an activation characteristic different from that of the mesophilic nuclease, and can be used with the mesophilic nuclease. Nucleic acid amplification complex treatment to be used.

Quantitative PCR using mesophilic nuclease and thermophilic proteinase is performed as follows:
1. (I) target cell material, (ii) one or more mesophilic nucleases that inactivate contaminating nucleic acids, and (iii) exhibit low activity against the amplification enzyme used in qPCR and extract target nucleic acids Preparing a reagent cocktail comprising a thermophilic proteinase and (iv) the reagents necessary to perform the qPCR (eg, dNTP's, buffer, MgCl ₂ , oligonucleotide primer, Taq DNA polymerase),
2. The reagent cocktail is incubated at 20 ° C. to 40 ° C. so that the mesophilic nuclease is activated for a period sufficient to cause degradation or deactivation of contaminating nucleic acids (note that at 20 ° C. to 40 ° C. Thermophilic proteinase activity is low and the mesophilic nuclease is not affected)
3. The reagent cocktail is incubated at 65 ° C. to 80 ° C. so as to activate the thermophilic proteinase (the thermophilic proteinase digests the mesophilic nuclease and simultaneously dissolves and degrades the cellular material to target nucleic acid. Causing the release of
4). Incubating the reagent cocktail at 90 ° C. to 95 ° C. to activate the Taq DNA polymerase and simultaneously deactivate the thermophilic proteinase;
5. The qPCR temperature cycling (amplification of the target nucleic acid) and detection are performed.

  As shown in the examples above, all steps of the process are temperature controlled and performed in a single closed tube or microtiter plate using a thermal cycler.

[Example 4: Apparatus provided with controlled light source]
FIG. 2 shows an embodiment of the above-described process combining inactivation of contaminating nucleic acid, nucleic acid extraction, and nucleic acid amplification performed using the apparatus. The device is a sealed unit, for example, activates ethidium monoazide (EMA), which is a nucleic acid inactivating reagent, and emits light having a wavelength of 510 nm capable of causing a covalent bond between EMA and contaminating nucleic acid. , A sealed unit provided with a light source for irradiating the 96-well microtiter plate. The purpose of the device is to (i) prevent the sample from being exposed to ambient light outside the device, (ii) prevent further contamination of the nucleic acid, and (iii) protect the user from strong light emitted from the light source The use of EMA in the protection room may be simplified.

  The apparatus of FIG. 2 has a size suitable for a 96-well microtiter plate, and a light-shielding chamber (or partially) provided with an array of light-emitting diodes (LEDs) that irradiate the 96-well microtiter plate with light of 510 nm wavelength. A light-shielding chamber having a light-shielding property, a timer that controls the incubation time and irradiation time in the dark, and a temperature control block that holds the 96-well microtiter plate at a desired temperature (Peltier element, thermoelectric cooler or TEC) Also known temperature control block).

  The advantage of using a light emitting diode of a specific wavelength is that it reduces the heating of the sample because less thermoelectricity is emitted, and it can reduce the heating of the sample because it uses less unnecessary wavelengths. The point which can use a power supply and the point which can suppress the photobleaching of a fluorescence reagent by use of the said low power supply. Furthermore, the use of a light emitting diode array ensures that the same amount of light is supplied to all wells of the 96 well microtiter plate.

  Also, the apparatus of FIG. 2 can be used, for example, to unexpectedly activate contaminating nucleic acids in qPCR reagents. The nucleic acid amplification process combined with other nucleic acid processes using ethidium monoazide or mesophilic nuclease as described in the above examples is used with the apparatus of FIG. The above device in conjunction with a nucleic acid inactivation reagent has particular advantages over qPCR.

  Photobleaching of fluorescent dies is a known problem in qPCR and has led to an investment in the development of more stable DNA binding dies for qPCR (Mao et al., 2007). The ability of the device to incorporate a contaminating nucleic acid inactivation step into qPCR allowed higher photostability of the qPCR DNA binding die, and ultimately higher detection sensitivity.

  Such a device is integrated as part of a thermal cycler or real-time PCR instrument, which can perform all steps of contaminating nucleic acid inactivation, target nucleic acid extraction, target nucleic acid amplification, and target nucleic acid detection One piece of hardware was created.

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It is the schematic of a composite nucleic acid detection process. A single chamber device provided with a light emitting diode array and used to inactivate contaminating nucleic acids, extract target nucleic acids, and amplify target nucleic acids using an encapsulating reagent. When subjected to DNA extraction and qPCR in a single vessel is a graph showing the C _T values obtained in qPCR reactions to various Escherichia coli cell counts. In this reaction treatment, no ethidium monoazide treatment was performed. Dashed line in the drawing shows a C _T values obtained when performing qPCR reactions against water only (cell number zero). The error bar is a standard deviation of ± 1 from the average value. When subjected to DNA extraction and qPCR in a single vessel is a graph showing the C _T values obtained in qPCR reactions to various Escherichia coli cell counts. In this reaction treatment, ethidium monoazide treatment was performed as part of the sequential reaction in the closed tube. Dashed line in the drawing shows a C _T values obtained when performing qPCR reactions against water only (cell number zero). The error bar is a standard deviation of ± 1 from the average value.

Claims (51)

A method for amplifying a target nucleic acid in a closed system, comprising:
i) an addition step of adding a nucleic acid inactivating reagent, a thermophilic proteinase, and a nucleic acid amplification reagent to a sample containing a target nucleic acid to constitute the closed system,
The nucleic acid inactivating reagent has an active form and an inactive form, and only the active form inactivates the contaminating nucleic acid in the closed system, thereby preventing the contaminating nucleic acid from reacting with the nucleic acid amplification reagent. ,
The nucleic acid inactivating reagent changes to the active form only when an external stimulus is applied,
The thermophilic proteinase is in an active form at about 65 ° C. to about 80 ° C. and an inactive form at about 90 ° C. or higher;
ii) a closing step for closing the closed system;
iii) an application step of applying the external stimulus for a time sufficient for the nucleic acid inactivating reagent to change from the inactive form to the active form,
The application prevents the contaminating nucleic acid in the closed system from reacting with the nucleic acid amplification reagent, and the nucleic acid inactivating reagent is applied after the contaminating nucleic acid stops reacting with the nucleic acid amplification reagent and then changes to an inactive form. Process,
iv) an incubation step of incubating the sample at about 65 ° C. to about 80 ° C., wherein the incubation activates a thermophilic proteinase and generates one or more of cell lysis, protein degradation, cell wall enzyme degradation, An incubation step in which target nucleic acids for amplification are extracted;
v) an incubation step of incubating the sample at about 90 ° C. or more, wherein the incubation causes a thermocatalytic proteinase autocatalytic reaction to occur;
vi) an amplification step of amplifying the target nucleic acid,
The closed system comprises a single tank or tube.   Before incubating the sample at about 65 ° C. to 80 ° C. or simultaneously with incubating the sample at about 65 ° C. to 80 ° C. The method of claim 1.   After the contaminating nucleic acid no longer reacts with the nucleic acid amplification reagent, the nucleic acid inactivation reagent is applied to the nucleic acid inactivation reagent by applying the external stimulus for a time sufficient for the nucleic acid inactivation reagent to change to the inactive form. 2. The method of claim 1, wherein the method is changed to an inactive form.   2. The method of claim 1, wherein after the contaminating nucleic acid no longer reacts with the nucleic acid amplification reagent, the nucleic acid inactivating reagent is changed to the inactive form by removing the external stimulus.   The nucleic acid inactivating reagent is prepared at a concentration sufficient to inactivate contaminating nucleic acid without inhibiting the amplification of the thermophilic proteinase, the nucleic acid amplification reagent, or the target nucleic acid. The method of claim 1.   The method according to claim 1, wherein the nucleic acid inactivating reagent is a mesophilic nucleic acid modifying enzyme.   The mesophilic nucleic acid modifying enzyme is selected from the group consisting of a double strand specific endonuclease, a double strand specific exonuclease, a single strand specific nuclease, a restriction endonuclease, RNAses, RNAseH, and an RNA modifying enzyme. The method according to claim 6.   7. The mesophilic nucleic acid modifying enzyme is activated by incubating at about 25 ° C. to 40 ° C. for a time sufficient to change the mesophilic nucleic acid modifying enzyme to the active form. The method described.   Inactivating the mesophilic nucleic acid modifying enzyme by incubating at about 65 ° C to 80 ° C for a time sufficient to change the mesophilic nucleic acid modifying enzyme to the inactive form. 6. The method according to 6.   The method according to claim 1, wherein the nucleic acid inactivating reagent is a nucleic acid intercalating agent.   The method according to claim 10, wherein when the external stimulus is applied, photolysis of the nucleic acid intercalating agent occurs, and the nucleic acid intercalating agent changes to the active form.   12. The method of claim 11, wherein the nucleic acid intercalator in the active form is covalently linked to a double stranded nucleic acid.   The method according to claim 10, wherein the nucleic acid intercalating agent is ethidium monoazide.   14. The method of claim 13, wherein the ethidium monoazide is provided at a concentration between about 1 [mu] g / ml and about 5 [mu] g / ml.   The method of claim 14, wherein the ethidium monoazide is provided at a concentration of about 3 μg / ml.   2. The method according to claim 1, wherein the external stimulus is a specific narrow spectrum wavelength light, a broad spectrum white light, or an ultraviolet light.   The external stimulus is a specific narrow spectrum wavelength light, a broad spectrum white light, or an ultraviolet light, sufficient to activate the nucleic acid insert by photolysis of the nucleic acid insert. The method according to claim 10.   2. The method of claim 1, wherein the external stimulus is sufficient thermal energy to activate the nucleic acid inactivating reagent without substantially activating the thermophilic proteinase.   The external stimulus is thermal energy, which increases the temperature of the closed system from about 25 ° C. to about 40 ° C. for a time sufficient to change the mesophilic nucleic acid modifying enzyme to the active form. The method of claim 18, wherein the thermal energy is sufficient.   2. The method of claim 1, wherein the thermophilic proteinase is EA1. (I) treating the sample with a mesophilic enzyme;
The method of claim 1, further comprising: (ii) incubating the sample at a temperature below about 40 ° C. for a time sufficient to remove cell walls from the cells.   The method according to claim 21, wherein the mesophilic enzyme is cellulase or lysozyme.   The method of claim 1, wherein amplification of the nucleic acid is detected by a fluorophore.   The method according to claim 1, wherein a PCR detection method is performed in the amplification step of the target nucleic acid.   The method according to claim 24, wherein the PCR detection method is selected from the group consisting of qPCR, multiplex PCR, and reverse transcription PCR.   The method according to claim 1, wherein an isothermal detection method is performed in the amplification step.   The isothermal detection method is selected from the group consisting of strand displacement amplification method, rolling circle amplification method, loop-mediated isothermal amplification method, chimeric primer-derived nucleic acid isothermal amplification method, Q-beta amplification system, and OneCutEventAmplificatioN 27. The method of claim 26.   The isothermal detection method includes nuclease chain reaction (NCR), RNAse-mediated nuclease chain reaction (RNCR), polymerase nuclease chain reaction (PNCR), RNAse-mediated detection (RMD), tandem repeat restriction enzyme promotion (TR-REF) chain reaction 27. The method of claim 26, wherein the method is selected from the group consisting of: an inverted reverse complement restriction enzyme promoted (IRC-REF) chain reaction.   The method according to claim 1, wherein SNP detection analysis is performed in the amplification step.   The method according to claim 1, wherein the nucleic acid amplification step is automated.   2. The method according to claim 1, wherein the nucleic acid amplification reagent is added using a microfluidic or solid dispenser.   The method according to claim 1, wherein the addition of the nucleic acid amplification reagent is performed using a microcapsule containing the nucleic acid amplification reagent.   33. The method of claim 32, wherein the microcapsules are pre-supplied to the vessel or tube.   The method according to claim 32, wherein the microcapsule is a heat-resistant capsule.   35. The method of claim 34, wherein the heat-resistant capsule is agarose or wax material beads.   37. The detection reagent is released from the heat-resistant capsule when the heat-sensitive capsule is exposed to a temperature sufficient to melt or decompose the heat-sensitive capsule. the method of.   The method according to claim 1, wherein the nucleic acid amplification reagent is resistant to proteolysis by the thermophilic proteinase.   2. The method of claim 1, wherein the sample is selected from the group consisting of blood, urine, saliva, semen, excrement, tissue, swab, tears, bones, teeth, hair, and mucus.   2. The method of claim 1, wherein the sample is selected from the group consisting of bacteria, fungi, archaea, eukaryotes, protozoa, and viruses.   40. The method of claim 39, wherein the sample is selected from the group consisting of salt water, fresh water, ice, soil, waste, and food.   The method of claim 1, wherein the vessel or tube is a device.   42. The method of claim 41, wherein the device is a handheld device.   42. The method of claim 41, wherein the device or a component of the device is disposable.   42. The method of claim 41, wherein the apparatus comprises an inlet, an outlet, a chamber, a detector for detecting the emitted fluorophore, and an excitation light source.   42. The method of claim 41, wherein the apparatus further comprises a microfluidic, a microchip, a nanopore technology, and a manipulator device.   42. The apparatus according to claim 41, comprising a sealed unit, a light shielding chamber, a nucleic acid inactivating reagent activation light source, a detector for detecting the emitted fluorophore, and an excitation light source. the method of.   47. The method of claim 46, wherein the nucleic acid inactivating reagent activating light source is selected from the group consisting of an incandescent lamp, a fluorescent lamp, and a light emitting diode array. A method for amplifying a target nucleic acid in a closed system, comprising:
i) an addition step of adding the mesophilic nucleic acid modifying enzyme, EA1 proteinase, and PCR amplification reagent to the sample containing the target nucleic acid to constitute the closed system,
The mesophilic nucleic acid modifying enzyme has an active form and an inactive form, and only the active form inactivates the contaminating nucleic acid in the closed system, thereby preventing the contaminating nucleic acid from reacting with the PCR amplification reagent,
The mesophilic nucleic acid modifying enzyme is in an active form at about 25 ° C to about 40 ° C, and is in an inactive form at about 65 ° C to about 80 ° C.
The EA1 proteinase is in an active form at about 65 ° C. to about 80 ° C. and an inactive form at about 90 ° C. or higher;
ii) a closing step for closing the closed system;
iii) an incubation step in which the sample is incubated at a temperature of about 25 ° C. to about 40 ° C. for a certain period of time, wherein the incubation activates the mesophilic nucleic acid modifying enzyme, and contaminated nucleic acid in the closed system reacts with the PCR amplification reagent. An incubating step to avoid,
iv) an incubation step in which the sample is incubated at a temperature of about 65 ° C. to about 80 ° C. for a certain period of time, wherein the incubation deactivates the mesophilic nucleic acid modifying enzyme, activates the EA1 proteinase, cell lysis, proteolysis, An incubation step in which target nucleic acids for amplification are extracted by the occurrence of one or more of the degradation of cell wall enzymes;
v) an incubation step of incubating the sample at about 90 ° C. or higher, wherein the incubation causes an autocatalytic reaction of the EA1 proteinase;
vi) an amplification step of amplifying the target nucleic acid,
The closed system comprises a single tank or tube. A method for amplifying a target nucleic acid in a closed system, comprising:
i) an addition step of adding ethidium monoazide, EA1 proteinase, and PCR amplification reagent to a sample containing the target nucleic acid to constitute the closed system,
The ethidium monoazide has an active form and an inactive form, and only the active form inactivates the contaminating nucleic acid in the closed system, thereby preventing the contaminating nucleic acid from reacting with the PCR amplification reagent,
The ethidium monoazide changes to the active form only when photolysis of ethidium monoazide occurs when narrow spectrum wavelength light, broad spectrum white light, or ultraviolet light is applied.
The EA1 proteinase is in an active form at about 65 ° C. to about 80 ° C. and an inactive form at about 90 ° C. or higher;
ii) a closing step for closing the closed system;
iii) an application step of applying the narrow spectrum wavelength light, broad spectrum white light, or ultraviolet light for a first time sufficient to change the ethidium monoazide from the inactive form to the active form. The irradiation prevents contaminating nucleic acids in the closed system from reacting with the nucleic acid amplification reagent, and the ethidium monoazide becomes inactive with respect to the subsequent nucleic acid by photolysis of the ethidium monoazide by the irradiation. Process,
iv) an incubation step in which the sample is incubated at a temperature of about 65 ° C. to about 80 ° C. for a second time, wherein the incubation activates the EA1 proteinase, and one of cell lysis, proteolysis, and cell wall enzyme degradation An incubation step in which the target nucleic acid for amplification is extracted by more than one occurrence;
v) an incubation step of incubating the sample at about 90 ° C. or higher, wherein the incubation causes an autocatalytic reaction of the EA1 proteinase;
vi) an amplification step of amplifying the target nucleic acid,
The closed system comprises a single tank or tube.   The method according to claim 1, wherein the amplification detection sensitivity is at least about twice or more the amplification detection sensitivity in the method not using the nucleic acid inactivating reagent.   The amplification detection sensitivity when the method according to claim 1 is carried out in a closed system is at least about twice the amplification detection sensitivity when the method according to claim 1 is carried out in an open system. The method of claim 1.

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