WO2022085530A1 - Procédé de prétraitement pour préparer un échantillon à fournir pour un procédé de détection d'acide nucléique étranger - Google Patents

Procédé de prétraitement pour préparer un échantillon à fournir pour un procédé de détection d'acide nucléique étranger Download PDF

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WO2022085530A1
WO2022085530A1 PCT/JP2021/037851 JP2021037851W WO2022085530A1 WO 2022085530 A1 WO2022085530 A1 WO 2022085530A1 JP 2021037851 W JP2021037851 W JP 2021037851W WO 2022085530 A1 WO2022085530 A1 WO 2022085530A1
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sample
nucleic acid
solution
aqueous solution
serine protease
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PCT/JP2021/037851
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Japanese (ja)
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洋介 川嶋
佳子 上倉
光生 前野
明生 杉山
広道 鈴木
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東洋紡株式会社
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Publication of WO2022085530A1 publication Critical patent/WO2022085530A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/11—Orthomyxoviridae, e.g. influenza virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/115—Paramyxoviridae, e.g. parainfluenza virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a pretreatment method for the preparation of a sample to be used in a method for detecting an exogenous nucleic acid. More specifically, the present invention relates to a method for quickly and easily extracting nucleic acid from a sample sample and preparing a sample suitable for subject to a nucleic acid amplification method, a nucleic acid detection method using the sample, and the like.
  • Detecting microorganisms contained in biological samples is important in clinical diagnosis and the like. Therefore, a method is performed in which nucleic acid in a biological sample is amplified by PCR or the like, the amplified nucleic acid is detected, and a microorganism is identified.
  • the biological sample contains various biological substances in addition to nucleic acid, if nucleic acid amplification is performed without purifying the biological sample, amplification inhibition and detection inhibition occur, and in most cases, correct measurement results are obtained. Cannot be obtained. Therefore, when detecting a microorganism contained in a biological sample, it is common to apply a pretreatment to the biological sample instead of using the biological sample as it is for detection.
  • nucleic acid purification capable of almost removing components other than nucleic acid is performed (Patent Document 1).
  • Patent Document 1 nucleic acid purification has problems that the operation is very complicated, the operation time is long, an organic solvent is used, and the reagent cost is high.
  • Non-Patent Document 1 a method of decomposing contaminants in a biological sample using proteinase K, which is a proteolytic enzyme, is also used.
  • Proteinase K is known to improve enzyme activity in the presence of some surfactants (Non-Patent Document 1).
  • the surfactant itself has an action of dissolving biological components such as proteins and lipids
  • Non-Patent Document 2 a method of treating a sample by using proteinase K and a surfactant in combination is also known.
  • a method of using a protein denaturing agent such as a chaotropic salt or urea instead of the surfactant (Patent Document 2).
  • nucleic acid amplification methods such as PCR
  • a large amount of surfactant or protein denaturing agent can denature DNA polymerase and significantly inhibit nucleic acid amplification.
  • proteinase K having enzyme activity remains in the sample after pretreatment
  • the DNA polymerase in the nucleic acid amplification reagent is decomposed by the action of proteinase K when the sample and the nucleic acid amplification reagent are mixed. Therefore, there is concern that nucleic acid amplification may be significantly inhibited.
  • an object of the present invention is to provide a method for preparing a sample capable of stable nucleic acid amplification by a simple pretreatment method using a specific proteolytic enzyme.
  • the present inventors have found that it is possible to provide an analytical sample suitable for the nucleic acid amplification method by adding a step of heating after treatment with serine protease and then adding an alkaline solution.
  • the present invention has been completed. That is, the outline of the present invention is as follows.
  • a method for pretreating a sample to be used in a method for detecting an exogenous nucleic acid which comprises the following steps (A) to (D): (A) Step of preparing a mixed solution containing a sample and a serine protease, (B) A step of performing an enzymatic reaction with a serine protease in the mixed solution prepared in the above step (A). (C) A step of heating the mixed solution subjected to the enzymatic reaction in the step (B), and (D) A step of mixing the mixed solution heat-treated in the step (C) with an alkaline solution.
  • the sample used in the step (A) is a biological sample, or at least one solvent selected from the group consisting of a biological sample and water, physiological saline, a liquid medium, and a buffer solution.
  • Item 2. The method according to Item 1, which is a mixture with and.
  • Item 3 The method according to Item 2, wherein the biological sample is a human-derived biological sample.
  • the buffer solution is at least one selected from the group consisting of Tris buffer solution, phosphate buffer solution, citrate phosphate buffer solution, phosphate buffered saline, Hanks solution, and Good Buffer. , Item 2 or 3.
  • [Item 5] The method according to any one of Items 1 to 4, wherein the enzymatic reaction in the step (B) is carried out by reacting the mixed solution at 50 to 70 ° C. for 1 to 10 minutes.
  • [Item 6] The method according to any one of Items 1 to 5, wherein the heating in the step (C) is a heat treatment at 90 to 100 ° C. for 1 to 10 minutes.
  • [Item 7] Item 1 to any one of Items 1 to 6, wherein the mixed solution heat-treated in the step (C) is cooled to a temperature lower than the reaction temperature in the step (B), and then the step (D) is performed. The method described.
  • [Item 8] The method according to any one of Items 1 to 7, wherein the serine protease is proteinase K.
  • the alkaline solution is an aqueous solution of potassium hydroxide, an aqueous solution of sodium hydroxide, an aqueous solution of lithium hydroxide, an aqueous solution of magnesium hydroxide, an aqueous solution of calcium hydroxide, an aqueous solution of barium hydroxide, an aqueous solution of potassium carbonate, an aqueous solution of sodium carbonate, an aqueous solution of magnesium carbonate, and the like.
  • the alkaline solution is an aqueous solution of potassium hydroxide, an aqueous solution of sodium hydroxide, an aqueous solution of lithium hydroxide, an aqueous solution of magnesium hydroxide, an aqueous solution of calcium hydroxide, an aqueous solution of barium hydroxide, an aqueous solution of potassium carbonate, an aqueous solution of sodium carbonate, an aqueous solution of magnesium carbonate, and the like.
  • [Item 11] The method according to any one of Items 1 to 10, wherein in the step (A), serine protease is mixed in a ratio of 1 ⁇ g to 4 ⁇ g with respect to 1 ⁇ L of the sample.
  • Item 12. The method according to any one of Items 1 to 11, wherein in the step (A), serine protease is mixed at a ratio of 0.03 U to 0.12 U with respect to 1 ⁇ L of the sample.
  • Item 6. The method according to any one of Items 1 to 12, wherein the exogenous nucleic acid is a nucleic acid of an enveloped virus.
  • [Item 14] From the group consisting of coronavirus family virus, orthomixovirus family virus, flavivirus family virus, togavirus family virus, bunyavirus family virus, paramyxovirus family virus, and phyllovirus family virus.
  • Item 6. The method according to Item 13, which is at least one selected.
  • Item 15] In a sample, which comprises a step of mixing a sample subjected to the pretreatment method according to any one of Items 1 to 14 with a nucleic acid amplification reagent and amplifying an exogenous nucleic acid that may be contained in the sample. A method for detecting an exogenous nucleic acid.
  • the method according to Item 15 further comprising the step of detecting the amplified nucleic acid.
  • Item 17 The method according to Item 16, wherein the detection step is performed by melting curve analysis.
  • the present invention it becomes possible to easily prepare a sample suitable for a nucleic acid detection method.
  • the prepared sample may be directly subjected to nucleic acid amplification without further nucleic acid purification treatment, and the foreign nucleic acid in the sample can be amplified and detected with good sensitivity.
  • the present invention is suitable, for example, as a method for pretreating a biological sample used for detecting foreign nucleic acids of microorganisms such as bacteria and viruses contained in the biological sample.
  • sample pretreatment method One of the embodiments of the present invention is a method of pretreating a sample to be used in a method for detecting an exogenous nucleic acid.
  • the method of the present invention is characterized by including at least the following steps (A) to (D).
  • C A step of heating the mixed solution subjected to the enzymatic reaction in the step (B), and
  • D A step of mixing the mixed solution heat-treated in the step (C) with an alkaline solution.
  • the sample in the step (A) can be any sample for which it is desired to confirm the presence or absence of foreign nucleic acid.
  • it may be a biological sample, an environmental sample, etc., but is not limited thereto.
  • a biological sample is preferred.
  • a sample collected from the living body may be used as it is, or a biological sample, a biological sample, water (for example, purified water, distilled water, ion-exchanged water, pure water, etc.), physiological saline, a liquid medium, and a biological sample.
  • a mixture such as a suspension or solution with at least one solvent selected from the group consisting of a buffer for example, a mixture of a biological sample and purified water, a mixture of a biological sample and a physiological saline solution, a biological sample and a liquid medium.
  • a buffer for example, a mixture of a biological sample and purified water, a mixture of a biological sample and a physiological saline solution, a biological sample and a liquid medium.
  • a mixture of a biological sample and a buffer solution, etc. can also be used as a biological sample.
  • the biological sample is not particularly limited as long as it is a sample derived from a living body, and examples thereof include animal and plant tissues, body fluids, excrement, and cells. More specifically, intraoral scrapes, pharyngeal swabs, nasal swabs, nasopharyngeal swabs, nasal aspirates, sputum, bronchial lavage fluids, alveolar lavage fluids, rectal wipes, vaginal secretions, cervical mucus, urinary tract Scraped material, stool suspension, blood, plasma, serum, blood culture solution, urine, saliva, sheep water, pus, mucus, pleural effusion, tissue section, skin, vomitus, feces, tympanic membrane incision, gastric lavage, intestinal lavage , Organ extract, tissue extract isolated culture colony, catheter lavage fluid, etc.
  • a preferred biological sample or suspension or solution thereof comprises a group consisting of intraoral scrapes, pharyngeal swabs, nasal swabs, nasopharyngeal swabs, nasal aspirates, sputum, saliva, bronchial lavage fluids, and alveolar lavage fluids. At least one selected.
  • the biological sample is, for example, a sample derived from a human or a non-human animal.
  • non-human animals include non-human mammals, such as dogs, cats, mice, rats, guinea pigs, hamsters, rabbits, pigs, cows, sheep and goats, and preferably dogs and cats. It is preferably a human-derived sample.
  • the method for collecting a sample from a living body is not particularly limited, and a known method can be used depending on the type, size, and purpose of the sample.
  • a collection method using a collection tool such as a cotton swab, a swab, a platinum loop, a dropper, a spatula, and a spoon.
  • the collected biological sample may be used as a liquid for the purpose of diluting or storing biological substances, for example. It can also be mixed with a liquid form such as a suspension.
  • a mixture such as a suspension or a solution is also used as a sample in the step (A).
  • the liquid medium is not particularly limited as long as it is a liquid medium.
  • LB medium bouillon medium, minimal medium, virus transport medium (VTM), general purpose transport medium (UTM), Ames medium, carry blare medium, Stuart medium, DMEM, MEM, or improved versions of these liquid media.
  • VTM virus transport medium
  • UDM general purpose transport medium
  • Ames medium carry blare medium, Stuart medium, DMEM, MEM, or improved versions of these liquid media.
  • LB medium bouillon medium
  • VTM virus transport medium
  • UDMEM general purpose transport medium
  • Ames medium carry blare medium
  • Stuart medium DMEM
  • MEM or improved versions of these liquid media.
  • the buffer solution is not particularly limited as long as it is a liquid having a buffering ability, and if it is generally used as a buffer solution in the fields of biochemistry and molecular biology, it can be used as a buffer solution in the present invention. can.
  • the buffer solution is preferably a Tris buffer solution, a phosphate buffer solution, a citrate phosphate buffer solution, a phosphate buffered saline solution, a Hanks solution, or a good buffer.
  • a simple operation before subjecting the sample to the step (A) of the present invention.
  • a simple operation centrifuge the biological sample to settle impurities and collect only the supernatant to prepare a sample for the pretreatment method of the present invention, or centrifuge the biological sample to leave only the lower layer or precipitate.
  • the sample components and the concentrated bacteria and viruses contained in the sample are used as the sample to be used for the pretreatment method of the present invention, and the biological sample is mixed with the liquid medium and / or the buffer solution and then centrifuged. Examples include, but are not limited to, collecting the supernatant, the lower layer, or the precipitate and using it as a sample to be used for the pretreatment method of the present invention.
  • the serine protease used in the present invention is a well-known proteolytic enzyme having a serine residue in the active center.
  • Serine proteases are classified into subtilisin-like serine proteases, chymotrypsin-like serine proteases and the like, all of which can be used in the present invention, but are preferably subtilisin-like serine proteases.
  • subtilisin-like serine protease for example, proteinase K can be mentioned as a preferable example, but the subtilisin-like serine protease is not limited thereto.
  • the proteinase K used in the present invention is a serine protease derived from Tritiracium album (Engyodontium album).
  • the proteinase K used in the present invention may be directly extracted from Tritiracium album, or may be produced by using a gene recombination technique (for example, recombinant from the same host cell or another host cell). It may be the one obtained by expressing it).
  • the properties of proteinase K used in the present invention are not particularly limited, and either liquid or solid can be used. It is preferably Proteinase K prepared in a liquid form.
  • proteinase K may be described interchangeably with protease K, Proteinase K or PK.
  • the step (A) in the present invention is a step of mixing the sample and the serine protease. This step prepares a mixed solution of the sample and serine protease.
  • the amount of serine protease used in the present invention is not particularly limited as long as the effects of the present invention can be achieved.
  • the ratio of the serine protease (for example, proteinase K) in step (A) to the sample is preferably mixed at a ratio of 0.01 ⁇ g to 15 ⁇ g of the serine protease to 1 ⁇ L of the sample.
  • the ratio of the serine protease (for example, proteinase K) in step (A) to the sample is such that the serine protease is 0.015 U to 0.3 U per 1 ⁇ L of the sample. It is better, and more preferably, the serine protease is mixed in a ratio of 0.03U to 0.12U.
  • the work of centrifuging the mixed solution of the sample and the serine protease and collecting the supernatant may be added.
  • the liquid portion can be recovered by the operation, and a part or all of the recovered liquid (mixed liquid) can be provided to the step (B).
  • the sample processing method of the present invention has an advantage that it is not always necessary to use a surfactant, a chaotropic salt, and urea in combination for the purpose of increasing the processing efficiency of the sample.
  • the use of a serine protease solution containing a small amount of surfactant for the purpose of storing or stabilizing serine protease is also within the scope of the present invention.
  • the step (B) in the present invention is a step of performing an enzymatic reaction with a serine protease.
  • this step some or all of the proteins in the sample are degraded by serine protease.
  • This step is usually carried out by heating to a temperature at which the serine protease used is active.
  • the temperature at the time of heating in this step is not particularly limited as long as the proteinase K can exert the enzyme activity, and is preferably 25 to 75 ° C., for example. Is 37 to 75 ° C, more preferably 50 to 70 ° C, and even more preferably 55 to 65 ° C.
  • the time for heating to the temperature is not particularly limited, but is preferably 1 to 15 minutes, more preferably 1 to 10 minutes, and even more preferably 3 to 10 minutes. Further, when heating is accompanied in the step (B), the temperature does not necessarily have to be fixed at a specific temperature, and may be changed as long as it is within the temperature range in which the serine protease can exert its activity.
  • the step (C) in the present invention is a step of heating the mixed solution subjected to the enzymatic reaction in the step (B). By heating in this step, some or all of the serine protease is inactivated, and some or all of the nucleolytic enzymes and proteolytic enzymes contained in the sample are inactivated, and the sample is inactivated. It is expected that some or all of the microorganisms and viruses will be killed. However, it is not necessary to kill all proteins, microorganisms, viruses, etc. in this step, and it is not required to measure whether enzyme inactivation or killing of microorganisms, etc. occurs in this step.
  • the heating temperature in this step is not particularly limited as long as it is higher than the temperature to which the mixed solution is exposed in step (B), but is preferably 80 to 100 ° C, more preferably 90 to 100 ° C, and further. It is preferably 95 to 100 ° C.
  • the heating time is not particularly limited, but is preferably 1 to 15 minutes, more preferably 1 to 10 minutes, and even more preferably 3 to 10 minutes. Further, the heating temperature in the step (C) does not necessarily have to be fixed at a specific temperature, and may be changed.
  • a step of cooling the mixed solution may be added after the step (C) is completed.
  • the cooling temperature is not particularly limited, but when heating is performed in the step (B), it is desirable to cool to a temperature lower than the heating temperature. For example, it is preferable to cool to a temperature lower than 55 ° C, preferably to a temperature lower than 50 ° C, more preferably to a temperature lower than 37 ° C, and even more preferably to a temperature lower than 25 ° C.
  • the cooling time here is not particularly limited, and may be, for example, 1 to 15 minutes, preferably 1 to 10 minutes, and more preferably 3 to 10 minutes.
  • the cooling method of this step is not particularly limited, and is allowed to stand at room temperature, stand in a refrigerator or freezer, stand on ice, cool using a temperature changing device such as an incubator or a water bath, and the like. It is possible in any way.
  • the alkaline solution used in step (D) in the present specification is not particularly limited as long as it is an alkaline solution.
  • the alkaline solution include potassium hydroxide aqueous solution, sodium hydroxide aqueous solution, lithium hydroxide aqueous solution, magnesium hydroxide aqueous solution, calcium hydroxide aqueous solution, barium hydroxide aqueous solution, potassium carbonate aqueous solution, sodium carbonate aqueous solution, magnesium carbonate aqueous solution, and calcium carbonate.
  • Examples thereof include an aqueous solution, which can be used alone or in combination of two or more.
  • the preferable alkaline solution is at least one selected from the group consisting of an aqueous solution of potassium hydroxide, an aqueous solution of sodium hydroxide, and an aqueous solution of lithium hydroxide. More preferred alkaline solutions are potassium hydroxide aqueous solution and / or sodium hydroxide aqueous solution.
  • the alkaline concentration is a volume molar concentration (M or mol / L), and an alkaline substance contained in 1 L of the alkaline solution (for example, an alkaline substance such as potassium hydroxide and / or sodium hydroxide as described above). It can be specified by the molar amount of).
  • the alkaline concentration in the mixed solution after the addition of the alkaline solution is not particularly limited as long as the effect of the present invention is exhibited.
  • the alkali concentration in the mixed solution after the addition of the alkaline solution in the step (D) is preferably, for example, 0.1 to 50 mM, preferably 0.5 to 0.5 mM. It is preferably 40 mM, more preferably 1 to 30 mM, still more preferably 5 to 30 mM, even more preferably 10 to 30 mM, particularly preferably 15 to 30 mM, and particularly preferably 20 to 30 mM.
  • the method of mixing the alkaline solution in the step (D) is not particularly limited, and the alkaline solution may be added to the container containing the mixed solution of the sample and the serine protease that have undergone the steps (A) to (C) with a micropipette or the like.
  • the alkaline solution may be dispensed into another container in advance and the mixed solution may be added thereto, and the order of addition thereof does not matter.
  • stirring may be performed after adding the alkaline solution.
  • the method is not particularly limited as long as the mixed solution and the alkaline solution can be sufficiently mixed, and overturning stirring, stirring with a vortex mixer, or the like is possible. Stirring conditions such as stirring strength and time can be appropriately set by those skilled in the art based on common general technical knowledge.
  • One of the embodiments of the present invention is to mix a sample pretreated by the method including the above steps (A) to (D) as a sample for analysis with a nucleic acid amplification reagent, and to obtain an exogenous nucleic acid that can be contained in the sample.
  • a method for detecting an exogenous nucleic acid in a sample which comprises a step of performing amplification.
  • mixing with a nucleic acid amplification reagent means further nucleic acid from a pretreated sample, such as nucleic acid purification with a column or removal of contaminants with an organic solvent after the step (D) is completed. It refers to mixing with a nucleic acid amplification reagent without performing a separation and purification step.
  • a step that does not involve separation and purification of nucleic acid such as transferring a part of the pretreated sample to another tube in an amount necessary for mixing with the nucleic acid amplification reagent, may be performed, and an embodiment including this step is also possible. It is within the scope of the present invention.
  • the exogenous nucleic acid refers to any nucleic acid that is not derived from the sample and is of unknown origin whether or not it can be normally contained in the sample.
  • the sample is a biological sample, it is a nucleic acid derived from a species other than the biological species from which the biological sample is derived.
  • nucleic acids of non-human species are referred to as exogenous nucleic acids.
  • the origin of the exogenous nucleic acid is not particularly limited, and examples thereof include bacteria, viruses, phages, fungi, parasites, and amoebas. Bacteria and viruses are more preferable, and viruses are more preferable.
  • the present invention is suitable for detecting such exogenous nucleic acid, and when the exogenous nucleic acid is detected by the method of the present invention, the sample contains the organism from which the exogenous nucleic acid is derived or the nucleic acid itself. It can mean that.
  • the present invention can be used in the detection of any exogenous nucleic acid, but is preferably suitable for the detection of viruses, especially enveloped viruses.
  • An enveloped virus is a virus in which genomic nucleic acid is encapsulated in an envelope composed of a lipid bilayer.
  • enveloped viruses include coronavirus family viruses (eg, SARS coronavirus, MERS coronavirus, SARS-CoV-2 coronavirus); orthomixoviridae viruses (eg, influenza A virus, influenza B virus, etc.).
  • Flavivirus family virus eg, hepatitis C virus, Japanese encephalitis virus, decavirus, pig fever virus
  • Togavirus family virus eg, ruin virus, Chikungunia virus
  • Rabdovir family virus eg) , Mad dog disease virus
  • Bunyavirus family virus eg, Crimea-Congo fever virus, Hunter virus
  • Paramyxovirus family virus eg, measles virus, human RS virus
  • Phyllovirus family virus eg, Ebola virus
  • the present invention is preferably detection of a coronavirus family virus or an orthomyxoviridae virus, and more preferably SARS coronavirus, MERS coronavirus, SARS-CoV-2. It is useful in detecting coronavirus, influenza A virus, influenza B virus, and influenza C virus.
  • a known nucleic acid amplification method can be used as the method used for nucleic acid amplification in the above method.
  • nucleic acid amplification methods include PCR method, LAMP method, LCR method, TMA method, SDA method, RT-PCR method, RT-LAMP method, NASBA method, TRC method, TMA method and the like. These techniques have already been established in the relevant technical field, and the method can be selected according to the purpose.
  • the nucleic acid amplification method performed in the present invention is preferably, but is not limited to, the PCR method (including the RT-PCR method) from the viewpoint that a higher effect can be more reliably obtained.
  • the nucleic acid amplification reagent used in the above method is not particularly limited as long as it is a reagent containing components necessary for nucleic acid amplification.
  • the contents may usually include DNA polymerases, nucleic acid primers, dNTPs, magnesium ions and the like.
  • the nucleic acid amplification reagent may be provided in a form in which one or a plurality of components are divided and encapsulated, and in that case, two or more reagents and the analysis sample are used in a mixed form. Be done.
  • the shape of the nucleic acid amplification reagent is not particularly limited, but it is preferably a liquid nucleic acid amplification reagent.
  • One of the embodiments of the present invention is a method in which the nucleic acid detection method further includes a step of detecting the amplified nucleic acid after nucleic acid amplification.
  • the mode of this detection step is not particularly limited, and can be carried out by any method known in the art. From the viewpoint that a higher effect can be more reliably obtained, the detection or melting curve analysis method by a real-time PCR method (including a real-time RT-PCR method) is preferable in the present invention. Further, in the detection, it is preferable to use a nucleic acid probe capable of specifically detecting the nucleic acid amplification product.
  • a probe labeled with a fluorescent quenching dye also referred to as a guanine quenching probe
  • fluorescent quenching dyes include 4,4-difluoro-5.7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), and carboxyrhodamine 6G.
  • a probe in which one end is labeled with a fluorescent substance and the other end is labeled with a quenching substance.
  • a fluorescent substance include rhodamine-based compounds; fluorosane-based compounds (for example, FAM (carboxyfluorescein)), and examples of the quenching substance include BHQ1 (BHQ: Black Hole Quencher (registered trademark)). ), BHQ2, BHQ3 and the like, but are not limited thereto.
  • a further embodiment of the present invention is a kit for performing a sample pretreatment method used in a method for detecting an exogenous nucleic acid including the steps (A) to (D).
  • the kit contains some or all of the reagents used in performing the steps (A)-(D), for example, one containing a serine protease and / or an alkaline solution, both of which are included. It is particularly preferable to include it.
  • Another embodiment of the present invention is for nucleic acid amplification for mixing a sample pretreated through the steps (A) to (D) with a nucleic acid amplification reagent as a sample for analysis and performing nucleic acid amplification.
  • a nucleic acid amplification reagent as a sample for analysis and performing nucleic acid amplification.
  • It is a kit for detecting a foreign nucleic acid in a sample containing a reagent.
  • the composition of the kit is not particularly limited as long as it is a kit containing the nucleic acid amplification reagent.
  • the nucleic acid amplification reagent may contain components necessary for detecting further amplified nucleic acid.
  • kits of may include, but is not limited to, for example, serine proteases, alkaline solutions, and / or nucleic acid amplification reagents, and may contain additional components as appropriate depending on the intended purpose.
  • the kit for performing the pretreatment method as described above, the kit for detecting the exogenous nucleic acid, and the kit for performing both of them are genes capable of detecting or quantifying the presence of a specific exogenous nucleic acid. It can optionally include a test reagent and / or an instruction manual or the like explaining how to use it.
  • the components of the nucleic acid amplification reagent are divided into two or more and enclosed in separate containers, or all the components are enclosed in one container, for example, packed in one package and used in the kit. It can be provided in an embodiment including information on the method. Further, a positive control solution or a negative control solution can also be included.
  • Example 1 Confirmation of the effect of the pretreatment method and the nucleic acid amplification method on the treated sample]
  • sample 30 ⁇ L of SARS-CoV-2 negative saliva sample, 55 ⁇ L of 10 mM Tris-HCl (pH 7.5), and 15 ⁇ L of NATtrol SARS-CoV-2 (recombinant) Stock (ZEPTOMETRIX) are mixed in a 1.5 mL tube, and simulated. 100 ⁇ L of SARS-CoV-2 positive sample was prepared.
  • the NATtrol SARS-CoV-2 (recombinant) Stock is equivalent to a sample in which the SARS-CoV-2 gene artificially synthesized in bacterial cells is encapsulated and imitates the enveloped virus SARS-CoV-2. do.
  • (2) Pretreatment method The pretreatment method of the present invention was performed on the above sample. Specifically, a mixed solution was prepared by adding a proteinase K solution (20 mg / mL, manufactured by Kanto Chemical Co., Inc.) to the sample in a PK amount, and heated at a temperature of T1 for M1 minutes. Then, it was heated at the temperature T2 for M2 minutes.
  • Reverse transcription, nucleic acid amplification and melting curve analysis 42 ° C for 2 minutes 97 ° C for 15 seconds (More than 1 cycle) 97 ° C for 1 second 58 ° C for 3 seconds 63 ° C for 5 seconds (More than 50 cycles) 94 ° C for 30 seconds 39 ° C for 30 seconds 40 ° C to 75 ° C (temperature rises at 0.09 ° C / sec)
  • FIG. 1 shows No. It is the result of having measured the pretreated analysis sample of 1.
  • a clear detection peak indicating the detection of SARS-CoV-2 has been obtained, and the SARS-CoV-2 Stock bacterial cells (sample imitating the envelope) mixed with the biological sample are treated by the pretreatment method of the present invention. It can be seen that the exposed SARS-CoV-2 gene was detected.
  • No. The detection results of 1 to 8 are shown in Table 2. All pretreated analytical samples were positive for SARS-CoV-2, with the amount of proteinase K, temperature T1, time M1, temperature T2, time M2, alkaline aqueous solution type, and alkaline concentration. It was clarified that the effect of the present invention can be obtained even if each element is changed.
  • Example 2 Nucleic acid detection from pretreated sample by real-time RT-PCR
  • (2) Real-time RT-PCR and determination Each of the above samples was added to the following reagents, and SARS-CoV-2 was detected under the following conditions. Rotor-Gene Q (QIAGEN) was used for real-time RT-PCR. The judgment was positive when the rising edge of the amplification curve was recognized and Ct was calculated.
  • QIAGEN Rotor-Gene Q
  • reagent A solution containing the following reagents was prepared.
  • 2XReaction Buffer TUNDERBIRD (R) PROBE One-Step qRT-PCR Kit, manufactured by Toyobo) 10 ⁇ L DNA Polymerase (same as above, manufactured by Toyobo) 1 ⁇ L RT Enzyme Mix (same as above, manufactured by Toyobo) 1 ⁇ L 10 ⁇ M Primer shown in SEQ ID NO: 1 1 ⁇ L 10 ⁇ M Primer shown in SEQ ID NO: 2 1 ⁇ L 10 ⁇ M Probe indicated by SEQ ID NO: 3 (labeled 5'end with FAM, 3'end labeled with BHQ1) 0.25 ⁇ L Purified water 1.75 ⁇ L Sample 5 ⁇ L
  • Real-time RT-PCR 50 ° C for 15 minutes 95 ° C for 2 minutes (More than 1 cycle) 95 ° C for 3 seconds Fluorescence detection at 55 ° C for 30 seconds (More than 45 cycles)
  • Result Table 3 is a table summarizing the results of real-time RT-PCR performed under the above conditions. No. Rising of the amplification curve was observed in all the pretreated analytical samples 1 to 8, and Ct was calculated. In addition, no amplification was observed in purified water, which was a negative control. From the above, it was confirmed that nucleic acids can be detected by real-time PCR or real-time RT-PCR from the sample treated by the pretreatment method of the present invention without the need for further nucleic acid purification treatment.
  • Example 3 Pretreatment of nasopharyngeal specimen
  • a biological sample was obtained by suspending a nasopharyngeal swab collected by a swab from a patient with SARS-CoV-2 infection in a general-purpose transport medium UTM (COPNA).
  • COPNA general-purpose transport medium UTM
  • a mixed solution was prepared by adding 10 ⁇ L (about 6 U) of a proteinase K solution (20 mg / mL, manufactured by Kanto Chemical Co., Inc.) to 100 ⁇ L of the biological sample. The mixture was heated at 65 ° C. for 5 minutes and then at 95 ° C. for 5 minutes.
  • Reverse transcription, nucleic acid amplification and melting curve analysis 42 ° C for 2 minutes 97 ° C for 15 seconds (More than 1 cycle) 97 ° C for 1 second 58 ° C for 3 seconds 63 ° C for 5 seconds (More than 50 cycles) 94 ° C for 30 seconds 39 ° C for 30 seconds 40 ° C to 75 ° C (temperature rises at 0.09 ° C / sec)
  • the pretreatment of the present invention is also effective for a sample derived from a clinical sample, and SARS-CoV-2 can be detected with high sensitivity from a nasopharyngeal sample, which is a biological sample, without requiring further nucleic acid purification. It has been shown.
  • Example 4 Pretreatment of sputum sample
  • the pretreatment method of the present invention it is possible to quickly and easily treat a biological sample and expose the foreign nucleic acid contained in the sample. Moreover, since nucleic acid amplification can be performed as it is on a measurement sample containing exposed foreign nucleic acid, rapid genetic testing becomes possible by using the present invention.

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Abstract

La présente invention a pour but de fournir un procédé simple pour préparer un échantillon à fournir pour un procédé de détection d'un acide nucléique étranger. La présente invention concerne un procédé de prétraitement pour un échantillon à fournir pour un procédé de détection d'acide nucléique étranger, le procédé comprenant les étapes suivantes : (a) une étape pour préparer une solution de mélange contenant un échantillon et une sérine protéase ; (b) une étape pour provoquer une réaction enzymatique par la sérine protéase dans la solution de mélange préparée à l'étape (a) ; (c) une étape pour chauffer la solution de mélange dans laquelle la réaction enzymatique a eu lieu à l'étape (B) ; et (d) une étape pour mélanger une solution alcaline et la solution de mélange qui a subi le chauffage à l'étape (c).
PCT/JP2021/037851 2020-10-19 2021-10-13 Procédé de prétraitement pour préparer un échantillon à fournir pour un procédé de détection d'acide nucléique étranger WO2022085530A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11332562A (ja) * 1998-04-28 1999-12-07 Ortho Clinical Diagnostics Inc 組織試料およびパラフィン包埋組織から核酸を抽出するための改良方法
JP2011188856A (ja) * 2010-03-11 2011-09-29 Samsung Techwin Co Ltd リアルタイムｐｃｒのための核酸テンプレートの製造

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11332562A (ja) * 1998-04-28 1999-12-07 Ortho Clinical Diagnostics Inc 組織試料およびパラフィン包埋組織から核酸を抽出するための改良方法
JP2011188856A (ja) * 2010-03-11 2011-09-29 Samsung Techwin Co Ltd リアルタイムｐｃｒのための核酸テンプレートの製造

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